Pub Date : 2025-01-01DOI: 10.1016/j.slasd.2024.100203
Tien T.T. Truong , Toan V. Phan , Yamin Oo , Oranart Matangkasombut , João N. Ferreira
{"title":"A novel platform for oral epithelia sheet biofabrication via magnetic 3D bioprinting","authors":"Tien T.T. Truong , Toan V. Phan , Yamin Oo , Oranart Matangkasombut , João N. Ferreira","doi":"10.1016/j.slasd.2024.100203","DOIUrl":"10.1016/j.slasd.2024.100203","url":null,"abstract":"","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"30 ","pages":"Article 100203"},"PeriodicalIF":2.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142831069","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 : 2024-12-21DOI: 10.1016/j.slasd.2024.100204
Zhongyao Ma , Bin Zou , Jiannan Zhao , Rui Zhang , Qiaoqiao Zhu , Xiaofeng Wang , Linan Xu , Xiang Gao , Xinyue Hu , Wei Feng , Wen Luo , Min Wang , Yunyun He , Zhifeng Yu , Weiren Cui , Qi Zhang , Letian Kuai , Wenji Su
To date, RNA-targeted chemical matter is under explored due to a lack of robust screening assays. In this study, we present a novel RNA-targeted small molecule screening approach using a specialized DNA-encoded library (DEL). Our findings reveal that the specialized DEL library, called “DEL Zipper”, can significantly reduce single-stranded DNA-RNA region interaction signals during various kinds of RNA selection. By performing the selection against both G-quadruplex, we have identified novel hits that interact with RNA targets and the results are validated through binding. This study demonstrates that the “DEL Zipper” method is a robust screening assay that has potential for discovering small molecule ligands for diverse RNA targets.
{"title":"Development of a DNA-encoded library screening method “DEL Zipper” to empower the study of RNA-targeted chemical matter","authors":"Zhongyao Ma , Bin Zou , Jiannan Zhao , Rui Zhang , Qiaoqiao Zhu , Xiaofeng Wang , Linan Xu , Xiang Gao , Xinyue Hu , Wei Feng , Wen Luo , Min Wang , Yunyun He , Zhifeng Yu , Weiren Cui , Qi Zhang , Letian Kuai , Wenji Su","doi":"10.1016/j.slasd.2024.100204","DOIUrl":"10.1016/j.slasd.2024.100204","url":null,"abstract":"<div><div>To date, RNA-targeted chemical matter is under explored due to a lack of robust screening assays. In this study, we present a novel RNA-targeted small molecule screening approach using a specialized DNA-encoded library (DEL). Our findings reveal that the specialized DEL library, called “DEL Zipper”, can significantly reduce single-stranded DNA-RNA region interaction signals during various kinds of RNA selection. By performing the selection against both G-quadruplex, we have identified novel hits that interact with RNA targets and the results are validated through binding. This study demonstrates that the “DEL Zipper” method is a robust screening assay that has potential for discovering small molecule ligands for diverse RNA targets.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100204"},"PeriodicalIF":2.7,"publicationDate":"2024-12-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142883990","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 : 2024-12-01DOI: 10.1016/j.slasd.2024.100198
Scott B. Ficarro , Zachary H. Marto , Nicholas M. Girardi , Dingyu Deng , Isabella Jaen Maisonet , Guillaume Adelmant , Laura E. Fleming , Mona Sharafi , Isidoro Tavares , Andrew Zhao , HyoJeon Kim , Hyuk-Soo Seo , Sirano Dhe-Paganon , Sara J. Buhrlage , Jarrod A. Marto
Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of compounds. Screening covalent fragments against purified protein targets reduces the demands on the mass spectrometer with respect to absolute throughput, detection limit, and dynamic range. Given these relaxed analytical requirements, we sought to develop an open-source, medium-throughput mass spectrometry system for target-based covalent fragment screening. Our platform comprises automated, dual LC desalting columns integrated with electrospray ionization for rapid sample introduction and mass spectrometry detection. The system is operated through a simple Python graphical user interface running on commodity microcontroller boards which allow integration with diverse liquid chromatography and mass spectrometry instruments. We provide scripts for fragment pooling, construction of sample batches, along with routines for data processing and visualization. The system enables primary screening of ∼10,000 covalent fragments per day in pooled format. In a proof-of-concept study we executed primary and secondary screens to identify 27 hit fragments against UCHL1, a deubiquitinating enzyme that is emerging as a drug target of interest across multiple clinical indications. We validated and triaged these covalent compounds through a series of orthogonal biochemical and chemoproteomic assays. The most promising chloroacetamide covalent fragment inhibited UCHL1 activity in vitro (IC50 < 5 µM) and exhibited dose-dependent binding along with good selectivity against 57 cellular DUBs as quantified by activity-based protein profiling.
{"title":"Open-source electrophilic fragment screening platform to identify chemical starting points for UCHL1 covalent inhibitors","authors":"Scott B. Ficarro , Zachary H. Marto , Nicholas M. Girardi , Dingyu Deng , Isabella Jaen Maisonet , Guillaume Adelmant , Laura E. Fleming , Mona Sharafi , Isidoro Tavares , Andrew Zhao , HyoJeon Kim , Hyuk-Soo Seo , Sirano Dhe-Paganon , Sara J. Buhrlage , Jarrod A. Marto","doi":"10.1016/j.slasd.2024.100198","DOIUrl":"10.1016/j.slasd.2024.100198","url":null,"abstract":"<div><div>Target-based screening of covalent fragment libraries with mass spectrometry has emerged as a powerful strategy to identify chemical starting points for small molecule inhibitors or find new binding pockets on proteins of interest. These libraries span diverse chemical space with a modest number of compounds. Screening covalent fragments against purified protein targets reduces the demands on the mass spectrometer with respect to absolute throughput, detection limit, and dynamic range. Given these relaxed analytical requirements, we sought to develop an open-source, medium-throughput mass spectrometry system for target-based covalent fragment screening. Our platform comprises automated, dual LC desalting columns integrated with electrospray ionization for rapid sample introduction and mass spectrometry detection. The system is operated through a simple Python graphical user interface running on commodity microcontroller boards which allow integration with diverse liquid chromatography and mass spectrometry instruments. We provide scripts for fragment pooling, construction of sample batches, along with routines for data processing and visualization. The system enables primary screening of ∼10,000 covalent fragments per day in pooled format. In a proof-of-concept study we executed primary and secondary screens to identify 27 hit fragments against UCHL1, a deubiquitinating enzyme that is emerging as a drug target of interest across multiple clinical indications. We validated and triaged these covalent compounds through a series of orthogonal biochemical and chemoproteomic assays. The most promising chloroacetamide covalent fragment inhibited UCHL1 activity in vitro (IC<sub>50</sub> < 5 µM) and exhibited dose-dependent binding along with good selectivity against 57 cellular DUBs as quantified by activity-based protein profiling.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100198"},"PeriodicalIF":2.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142775432","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 : 2024-12-01DOI: 10.1016/j.slasd.2024.100197
Mohamed Ismail , Gareth Davies , Graham Sproat , Tiziana Monteverde , Jonathan Tart , Marta Acebrón-García-de-Eulate , Andrea Gohlke , David Hancock , Santosh Adhikari , Sandra Stefanovic-Barrett , David M Smith , Vikki Flemington , Emma S. Gleave-Hanford , Geoffrey A. Holdgate , Jason G. Kettle , Julian Downward
The NanoBiT Biochemical Assay (NBBA) was designed as a biochemical format of the NanoBiT cellular assay, aiming to screen weak protein-protein interactions (PPIs) in mammalian cell lysates. Here we present a High Throughput Screening (HTS) application of the NBBA to screen small molecule and fragment libraries to identify compounds that block the interaction of KRAS-G12D with phosphatidylinositol 3-kinase (PI3K) p110α. This interaction promotes PI3K activity, resulting in the promotion of cell growth, proliferation and survival, and is required for tumour initiation and growth in mouse lung cancer models, whilst having little effect on the health of normal adult mice, establishing the significance of the p110α/KRAS interaction as an oncology drug target. Despite the weak binding affinity of the p110α/KRAS interaction (KD = 3 μM), the NBBA proved to be robust and displayed excellent Z’-factor statistics during the HTS primary screening of 726,000 compounds, which led to the identification of 8,000 active compounds. A concentration response screen comparing KRAS/p110α with two closely related PI3K isoforms, p110δ and p110γ, identified selective p110α-specific compounds and enabled derivation of an IC50 for these hits. We identified around 30 compounds showing greater than 20-fold selectivity towards p110α versus p110δ and p110γ with IC50 < 2 μM. By using Differential Scanning Fluorimetry (DSF) we confirmed several compounds that bind directly to purified p110α. The most potent hits will be followed up by co-crystallization with p110α to aid further elucidation of the nature of the interaction and extended optimisation of these compounds.
{"title":"High throughput application of the NanoBiT Biochemical Assay for the discovery of selective inhibitors of the interaction of PI3K-p110α with KRAS","authors":"Mohamed Ismail , Gareth Davies , Graham Sproat , Tiziana Monteverde , Jonathan Tart , Marta Acebrón-García-de-Eulate , Andrea Gohlke , David Hancock , Santosh Adhikari , Sandra Stefanovic-Barrett , David M Smith , Vikki Flemington , Emma S. Gleave-Hanford , Geoffrey A. Holdgate , Jason G. Kettle , Julian Downward","doi":"10.1016/j.slasd.2024.100197","DOIUrl":"10.1016/j.slasd.2024.100197","url":null,"abstract":"<div><div>The NanoBiT Biochemical Assay (NBBA) was designed as a biochemical format of the NanoBiT cellular assay, aiming to screen weak protein-protein interactions (PPIs) in mammalian cell lysates. Here we present a High Throughput Screening (HTS) application of the NBBA to screen small molecule and fragment libraries to identify compounds that block the interaction of KRAS-G12D with phosphatidylinositol 3-kinase (PI3K) p110α. This interaction promotes PI3K activity, resulting in the promotion of cell growth, proliferation and survival, and is required for tumour initiation and growth in mouse lung cancer models, whilst having little effect on the health of normal adult mice, establishing the significance of the p110α/KRAS interaction as an oncology drug target. Despite the weak binding affinity of the p110α/KRAS interaction (K<sub>D</sub> = 3 μM), the NBBA proved to be robust and displayed excellent Z’-factor statistics during the HTS primary screening of 726,000 compounds, which led to the identification of 8,000 active compounds. A concentration response screen comparing KRAS/p110α with two closely related PI3K isoforms, p110δ and p110γ, identified selective p110α-specific compounds and enabled derivation of an IC<sub>50</sub> for these hits. We identified around 30 compounds showing greater than 20-fold selectivity towards p110α versus p110δ and p110γ with IC<sub>50</sub> < 2 μM. By using Differential Scanning Fluorimetry (DSF) we confirmed several compounds that bind directly to purified p110α. The most potent hits will be followed up by co-crystallization with p110α to aid further elucidation of the nature of the interaction and extended optimisation of these compounds.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100197"},"PeriodicalIF":2.7,"publicationDate":"2024-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142755972","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 : 2024-11-17DOI: 10.1016/j.slasd.2024.100195
Olga Antipova , Valeria Moiseenko , Fatima Dzarieva , Ekaterina Savchenko , Igor Pronin , Galina Pavlova , Alexey Kopylov
Development of aptatheranostics for glioblastoma (GB) requires investigating aptamer interactions with cells. The paper has described flow cytometry (FC) assessment of direct interactions of fluorescent anti-CD133 aptamers with cells, focusing on cell cultures derived from patient GB (CCPGB). Conventional cell lines with different levels of CD133 mRNA, Caco-2 and HCT116, were used to compare interactions with known 2′FY-RNA aptamer A15 and DNA aptamers of Ap and Cs series, labeled with FAM and Cy5. In addition, interactions of certain non-aptameric oligonucleotides were studied. In the case of antibody interactions with cells, FC signals, mean fluorescence intensities (MFIs), correlated with sizable amounts of CD133 mRNA in Caco-2 cells, and CCPGBs 107 and G01. Unexpectedly, MFI per se could not be the solid indicator of specific interactions of aptamer - CD133/cell. Instead, two types of interactions, target CD133-driven and off-target membrane-associated ones, contribute to MFI. The latter was notably observed for CCPGB Sus/fP2 with tiny CD133 mRNA amount. To prove specificity of aptamer - CD133/cell interactions, titration experiments have been performed, revealing half-saturation concentrations of 120±27 for 2′FY-RNA A15 and 180±12 for DNA Cs5 with Caco-2 cells. This knowledge is an essential step to develop aptatheranostics for GB.
{"title":"Varieties of interactions of anti-CD133 aptamers with cell cultures from patient glioblastoma","authors":"Olga Antipova , Valeria Moiseenko , Fatima Dzarieva , Ekaterina Savchenko , Igor Pronin , Galina Pavlova , Alexey Kopylov","doi":"10.1016/j.slasd.2024.100195","DOIUrl":"10.1016/j.slasd.2024.100195","url":null,"abstract":"<div><div>Development of aptatheranostics for glioblastoma (GB) requires investigating aptamer interactions with cells. The paper has described flow cytometry (FC) assessment of direct interactions of fluorescent anti-CD133 aptamers with cells, focusing on cell cultures derived from patient GB (CCPGB). Conventional cell lines with different levels of CD133 mRNA, Caco-2 and HCT116, were used to compare interactions with known 2′FY-RNA aptamer A15 and DNA aptamers of Ap and Cs series, labeled with FAM and Cy5. In addition, interactions of certain non-aptameric oligonucleotides were studied. In the case of antibody interactions with cells, FC signals, mean fluorescence intensities (MFIs), correlated with sizable amounts of CD133 mRNA in Caco-2 cells, and CCPGBs 107 and G01. Unexpectedly, MFI <em>per se</em> could not be the solid indicator of specific interactions of aptamer - CD133/cell. Instead, two types of interactions, target CD133-driven and off-target membrane-associated ones, contribute to MFI. The latter was notably observed for CCPGB Sus/fP2 with tiny CD133 mRNA amount. To prove specificity of aptamer - CD133/cell interactions, titration experiments have been performed, revealing half-saturation concentrations of 120±27 for 2′FY-RNA A15 and 180±12 for DNA Cs5 with Caco-2 cells. This knowledge is an essential step to develop aptatheranostics for GB.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100195"},"PeriodicalIF":2.7,"publicationDate":"2024-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142649938","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}
Targeting transforming growth factor-β (TGF-β) receptors is a promising pharmacological approach to normalize aberrant signaling in genetic and non-genetic TGF-β associated diseases including fibrosis, cancer, cardiovascular and musculoskeletal disorders. To identify novel TGF-β receptor kinase inhibitors, methods like in vitro kinase assays, western blot or transcriptional reporter assays are often used for screening purposes. While these methods may have certain advantages, the lack of integration of key features such as receptor specificity, high-throughput capability, and cellular context resemblance remains a major disadvantage. This deficiency could ultimately hinder the translation of study outcomes into later (clinical) stages of drug development. In this study, we introduce an adjusted and optimized live cell NanoBRET Target Engagement (TE)-based method to identify TGF-β receptor specific kinase inhibitors. This comprehensive toolkit contains various TGF-β type I and type II receptors, with corresponding nanoBRET tracers, and disease-related cell lines, including novel non-commercially available materials. The nanoBRET capacity and kinase inhibitory window can be significantly enhanced for functional measurements when stable expression cell lines and substantially low tracer concentrations are used. In addition, this system can be tailored to study TGF-β associated genetic disorders and possibly be used to screen for disease-specific therapeutics. Therefore, the use of this optimized, live cell, antibody-independent nanoBRET Target Engagement assay is highly encouraged for future high-throughput compound screens targeting TGF-β/BMP receptors.
{"title":"TGF-β receptor-specific NanoBRET Target Engagement in living cells for high-throughput kinase inhibitor screens","authors":"Marius Wits , Nicole Haarmans , Gonzalo Sanchez-Duffhues , Marie-José Goumans","doi":"10.1016/j.slasd.2024.100196","DOIUrl":"10.1016/j.slasd.2024.100196","url":null,"abstract":"<div><div>Targeting transforming growth factor-β (TGF-β) receptors is a promising pharmacological approach to normalize aberrant signaling in genetic and non-genetic TGF-β associated diseases including fibrosis, cancer, cardiovascular and musculoskeletal disorders. To identify novel TGF-β receptor kinase inhibitors, methods like in vitro kinase assays, western blot or transcriptional reporter assays are often used for screening purposes. While these methods may have certain advantages, the lack of integration of key features such as receptor specificity, high-throughput capability, and cellular context resemblance remains a major disadvantage. This deficiency could ultimately hinder the translation of study outcomes into later (clinical) stages of drug development. In this study, we introduce an adjusted and optimized live cell NanoBRET Target Engagement (TE)-based method to identify TGF-β receptor specific kinase inhibitors. This comprehensive toolkit contains various TGF-β type I and type II receptors, with corresponding nanoBRET tracers, and disease-related cell lines, including novel non-commercially available materials. The nanoBRET capacity and kinase inhibitory window can be significantly enhanced for functional measurements when stable expression cell lines and substantially low tracer concentrations are used. In addition, this system can be tailored to study TGF-β associated genetic disorders and possibly be used to screen for disease-specific therapeutics. Therefore, the use of this optimized, live cell, antibody-independent nanoBRET Target Engagement assay is highly encouraged for future high-throughput compound screens targeting TGF-β/BMP receptors.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100196"},"PeriodicalIF":2.7,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142634383","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}
{"title":"The history, landscape, and outlook of human cell line authentication and security","authors":"Elijah Harbut , Yiorgos Makris , Alexander Pertsemlidis , Leonidas Bleris","doi":"10.1016/j.slasd.2024.100194","DOIUrl":"10.1016/j.slasd.2024.100194","url":null,"abstract":"","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 8","pages":"Article 100194"},"PeriodicalIF":2.7,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142634405","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 : 2024-11-08DOI: 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}
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