Pub Date : 2025-06-24DOI: 10.1016/j.slasd.2025.100245
Charles S. Lay, Elvira Diamantopoulou, Katharina L. Dürr, Idlir Liko, Steven J. Charlton
The generation of action potentials in neuronal cells and many other physiological processes involve the transport of chloride ions. Whilst there have been advances in chloride imaging techniques utilizing FRET biosensors, there is a lack of methodologies that are amenable to high-throughput screening for drug discovery. In this study, we developed a novel BRET-based biosensor (Glorider), utilizing a chloride-sensitive GFP variant fused to NanoLuciferase. The Glorider biosensor was then used to kinetically measure the effect of WNK, KCC2 and NKCC1 modulators in real time in living cells, including recently reported KCC2 agonists.
{"title":"Development of a BRET based chloride biosensor for high throughput screening of KCC2 modulators","authors":"Charles S. Lay, Elvira Diamantopoulou, Katharina L. Dürr, Idlir Liko, Steven J. Charlton","doi":"10.1016/j.slasd.2025.100245","DOIUrl":"10.1016/j.slasd.2025.100245","url":null,"abstract":"<div><div>The generation of action potentials in neuronal cells and many other physiological processes involve the transport of chloride ions. Whilst there have been advances in chloride imaging techniques utilizing FRET biosensors, there is a lack of methodologies that are amenable to high-throughput screening for drug discovery. In this study, we developed a novel BRET-based biosensor (Glorider), utilizing a chloride-sensitive GFP variant fused to NanoLuciferase. The Glorider biosensor was then used to kinetically measure the effect of WNK, KCC2 and NKCC1 modulators in real time in living cells, including recently reported KCC2 agonists.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100245"},"PeriodicalIF":2.7,"publicationDate":"2025-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144501316","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 : 2025-06-21DOI: 10.1016/j.slasd.2025.100244
Zhi-Bin Tong, Srilatha Sakamuru, James Travers, Tuan Xu, Shu Yang, Menghang Xia, Anton Simeonov, Ruili Huang, David Gerhold
Although multiple pesticides and solvents are risk factors for Parkinson’s disease [1] and other neurodegenerative diseases, most risk factors remain undiscovered. We previously identified the metallothionein gene MT1G as a biomarker for neurotoxicity induced by all seven neurotoxicants tested in LUHMES dopaminergic neurons. Here we used CRISP/R technology to insert a HiBiT tag into the MT1G gene of the LUHMES cell line. The engineered LUHMES MT1G::HiBiT cell lines were used to develop a quantitative high throughput screening [2] assay in a 3D-suspension culture platform with 1536 well microplates. We validated this qHTS assay by screening the LOPAC (Library of Pharmacologically Active Compounds) collection composed of 1280 compounds plus 88 selected Tox21 chemicals, demonstrating high signal-to-noise and reproducibility. In screening this library, 49 compounds were confirmed to significantly increase MT1G-HiBiT activity, including 35 compounds that exhibited cytotoxicity below 50 μM, and 14 noncytotoxic compounds. Most of these MT1G-HiBiT inducers killed cells at concentrations moderately higher than their MT1G-HiBiT activation potencies (AC50), however 14 showed MT1G-HiBiT AC50 values more than 3-fold lower than cytotoxicity IC50 values, and two showed higher values. Among the 49 MT1G-HiBiT inducers, 45 compounds resembled chelators. To test this apparent association, 27 known chelators were gathered and tested. Of these, 23 were active in the MT1G-HiBiT activity assay, confirming the propensity of chelators to activate MT1G transcription. Screening chemical libraries with this validated assay and characterizing the effects of active chemicals on cultured neurons may enable the identification of neurotoxicants or neurotoxic chemotypes that may cause neurodegenerative diseases.
{"title":"MT1G activation in dopaminergic neurons identifies chelators and their relationships to cytotoxicity","authors":"Zhi-Bin Tong, Srilatha Sakamuru, James Travers, Tuan Xu, Shu Yang, Menghang Xia, Anton Simeonov, Ruili Huang, David Gerhold","doi":"10.1016/j.slasd.2025.100244","DOIUrl":"10.1016/j.slasd.2025.100244","url":null,"abstract":"<div><div>Although multiple pesticides and solvents are risk factors for Parkinson’s disease [<span><span>1</span></span>] and other neurodegenerative diseases, most risk factors remain undiscovered. We previously identified the metallothionein gene <em>MT1G</em> as a biomarker for neurotoxicity induced by all seven neurotoxicants tested in LUHMES dopaminergic neurons. Here we used CRISP/R technology to insert a HiBiT tag into the <em>MT1G</em> gene of the LUHMES cell line. The engineered LUHMES <em>MT1G</em>::HiBiT cell lines were used to develop a quantitative high throughput screening [<span><span>2</span></span>] assay in a 3D-suspension culture platform with 1536 well microplates. We validated this qHTS assay by screening the LOPAC (Library of Pharmacologically Active Compounds) collection composed of 1280 compounds plus 88 selected Tox21 chemicals, demonstrating high signal-to-noise and reproducibility. In screening this library, 49 compounds were confirmed to significantly increase MT1G-HiBiT activity, including 35 compounds that exhibited cytotoxicity below 50 μM, and 14 noncytotoxic compounds. Most of these MT1G-HiBiT inducers killed cells at concentrations moderately higher than their MT1G-HiBiT activation potencies (AC<sub>50</sub>), however 14 showed MT1G-HiBiT AC<sub>50</sub> values more than 3-fold lower than cytotoxicity IC<sub>50</sub> values, and two showed higher values. Among the 49 MT1G-HiBiT inducers, 45 compounds resembled chelators. To test this apparent association, 27 known chelators were gathered and tested. Of these, 23 were active in the MT1G-HiBiT activity assay, confirming the propensity of chelators to activate <em>MT1G</em> transcription. Screening chemical libraries with this validated assay and characterizing the effects of active chemicals on cultured neurons may enable the identification of neurotoxicants or neurotoxic chemotypes that may cause neurodegenerative diseases.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100244"},"PeriodicalIF":2.7,"publicationDate":"2025-06-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144478192","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 : 2025-06-16DOI: 10.1016/j.slasd.2025.100243
Lan Mi , Mingxu You
Fluorescence- and bioluminescence-based probes are valuable tools for understanding cell functions in health and disease. Bioluminescence offers an ideal complementary readout to fluorescence due to its minimal background interference and self-illuminating nature. We previously introduced the first type of genetically encodable RNA-based bioluminescence resonance energy transfer (BRET) sensors. These RNA-based probes are highly programmable and can be modularly engineered to detect various cellular targets. While this system was successfully validated in vitro and from the entire cell population within a microplate, the BRET signals were quite dim and difficult to visualize at the single-cell level under a microscope. The ability of single-cell bioluminescence imaging is critical for studying cell-to-cell variations and spatiotemporal changes of cellular targets in different signaling pathways or upon drug treatment. In this study, we will introduce strategies that can enhance the functionality and capability of RNA-based BRET sensors for real-time cellular imaging and sensing. Using commonly used widefield microscopes, single-cell bioluminescent detection of various metabolites and other small molecules can be achieved in both bacterial and mammalian cells. This advancement represents a significant step toward the future development of genetically encoded RNA-based bioluminescent tools for studying disease mechanisms, high-throughput drug screening, and in vivo imaging.
{"title":"Genetically encoded fluorogenic RNA-based bioluminescence resonance energy transfer (BRET) sensors for cellular imaging and target detection","authors":"Lan Mi , Mingxu You","doi":"10.1016/j.slasd.2025.100243","DOIUrl":"10.1016/j.slasd.2025.100243","url":null,"abstract":"<div><div>Fluorescence- and bioluminescence-based probes are valuable tools for understanding cell functions in health and disease. Bioluminescence offers an ideal complementary readout to fluorescence due to its minimal background interference and self-illuminating nature. We previously introduced the first type of genetically encodable RNA-based bioluminescence resonance energy transfer (BRET) sensors. These RNA-based probes are highly programmable and can be modularly engineered to detect various cellular targets. While this system was successfully validated <em>in vitro</em> and from the entire cell population within a microplate, the BRET signals were quite dim and difficult to visualize at the single-cell level under a microscope. The ability of single-cell bioluminescence imaging is critical for studying cell-to-cell variations and spatiotemporal changes of cellular targets in different signaling pathways or upon drug treatment. In this study, we will introduce strategies that can enhance the functionality and capability of RNA-based BRET sensors for real-time cellular imaging and sensing. Using commonly used widefield microscopes, single-cell bioluminescent detection of various metabolites and other small molecules can be achieved in both bacterial and mammalian cells. This advancement represents a significant step toward the future development of genetically encoded RNA-based bioluminescent tools for studying disease mechanisms, high-throughput drug screening, and <em>in vivo</em> imaging.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100243"},"PeriodicalIF":2.7,"publicationDate":"2025-06-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144328043","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}
Drug discovery and development have been a significant focus of medicinal and pharmaceutical research, continually striving to meet the growing challenges posed by complex diseases and medical conditions. In drug development, quantum dots (QDs) can be utilized in fluorescent assays for drug discovery and as fluorescent labels in drug delivery systems to monitor the metabolism of drugs in the body. As efforts to unravel the mysteries of human health and design innovative therapeutic solutions increase, the roles of model organisms in advancing understanding and accelerating discovery and development are also expanding. Zebrafish (Danio rerio) have emerged as a prominent model organism in the field of drug screening and development due to their unique biological attributes and experimental advantages. Many pharmaceutical products and drugs developed in the pharmaceutical industry fail in clinical trials due to unanticipated toxic side effects. Similarly, despite the interesting characteristics and versatile applications of QDs in drug development, there are a limited number of clinical trials involving QDs, hindered by complex pharmaceutical, industrial, technical, and biological challenges such as toxicity. Therefore, this article aims to highlight the importance of using zebrafish embryos and eleutheroembryos models for the toxicological assessment of pharmaceutical drugs and QDs in drug delivery and development. This review summarizes the developments available in the literature regarding the evaluation of the toxicity of QDs and drugs using zebrafish assays. The use of zebrafish models for safety profiling and pharmacological preclinical screening of pharmaceutical drugs and QDs will provide more insights than cellular assays and offer valuable information for mammalian experiments.
{"title":"Toxicity evaluation of pharmaceutical drugs and quantum dots (QDs) using zebrafish embryos – A comprehensive review","authors":"Motunrayo Faderera Adegoke , Olamide Abiodun Daramola , Kayode Omotayo Adeniyi , Madan Poka , Patrick Hulisani Demana , Xavier Siwe Noundou","doi":"10.1016/j.slasd.2025.100241","DOIUrl":"10.1016/j.slasd.2025.100241","url":null,"abstract":"<div><div>Drug discovery and development have been a significant focus of medicinal and pharmaceutical research, continually striving to meet the growing challenges posed by complex diseases and medical conditions. In drug development, quantum dots (QDs) can be utilized in fluorescent assays for drug discovery and as fluorescent labels in drug delivery systems to monitor the metabolism of drugs in the body. As efforts to unravel the mysteries of human health and design innovative therapeutic solutions increase, the roles of model organisms in advancing understanding and accelerating discovery and development are also expanding. Zebrafish (Danio rerio) have emerged as a prominent model organism in the field of drug screening and development due to their unique biological attributes and experimental advantages. Many pharmaceutical products and drugs developed in the pharmaceutical industry fail in clinical trials due to unanticipated toxic side effects. Similarly, despite the interesting characteristics and versatile applications of QDs in drug development, there are a limited number of clinical trials involving QDs, hindered by complex pharmaceutical, industrial, technical, and biological challenges such as toxicity. Therefore, this article aims to highlight the importance of using zebrafish embryos and eleutheroembryos models for the toxicological assessment of pharmaceutical drugs and QDs in drug delivery and development. This review summarizes the developments available in the literature regarding the evaluation of the toxicity of QDs and drugs using zebrafish assays. The use of zebrafish models for safety profiling and pharmacological preclinical screening of pharmaceutical drugs and QDs will provide more insights than cellular assays and offer valuable information for mammalian experiments.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100241"},"PeriodicalIF":2.7,"publicationDate":"2025-06-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144230301","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 : 2025-05-18DOI: 10.1016/j.slasd.2025.100240
Somaya A. Abdel-Rahman , Moustafa T. Gabr
The SLIT2/ROBO1 signaling axis plays a critical role in cell migration, angiogenesis, and immune regulation, contributing to tumor progression, metastasis, and therapy resistance. SLIT2 is highly expressed in various malignancies, where it promotes immune evasion by recruiting tumor-associated macrophages and disrupting vascular integrity, ultimately diminishing therapeutic efficacy. Beyond cancer, SLIT2/ROBO1 is implicated in neural development, fibrosis, and vascular remodeling, making it a potential but underexplored therapeutic target. However, no small-molecule inhibitors of SLIT2/ROBO1 interaction currently exist. Herein, we describe the development and optimization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for high-throughput screening of small-molecule inhibitors targeting this pathway. Using recombinant SLIT2 and ROBO1, we established a robust assay that enables high-throughput screening (HTS) of chemical libraries of small molecules for SLIT2/ROBO1 inhibition. Screening a focused chemical library of protein-protein interaction (PPI) inhibitors identified SMIFH2 as a SLIT2/ROBO1 inhibitor, demonstrating its ability to disrupt the interaction in a dose-dependent manner. Our study introduces a novel screening platform for identifying small molecule inhibitors of SLIT2/ROBO1, laying the foundation for future drug discovery efforts aimed at targeting this signaling axis in cancer and other diseases.
{"title":"Optimization and development of a high-throughput TR-FRET screening assay for SLIT2/ROBO1 interaction","authors":"Somaya A. Abdel-Rahman , Moustafa T. Gabr","doi":"10.1016/j.slasd.2025.100240","DOIUrl":"10.1016/j.slasd.2025.100240","url":null,"abstract":"<div><div>The SLIT2/ROBO1 signaling axis plays a critical role in cell migration, angiogenesis, and immune regulation, contributing to tumor progression, metastasis, and therapy resistance. SLIT2 is highly expressed in various malignancies, where it promotes immune evasion by recruiting tumor-associated macrophages and disrupting vascular integrity, ultimately diminishing therapeutic efficacy. Beyond cancer, SLIT2/ROBO1 is implicated in neural development, fibrosis, and vascular remodeling, making it a potential but underexplored therapeutic target. However, no small-molecule inhibitors of SLIT2/ROBO1 interaction currently exist. Herein, we describe the development and optimization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay for high-throughput screening of small-molecule inhibitors targeting this pathway. Using recombinant SLIT2 and ROBO1, we established a robust assay that enables high-throughput screening (HTS) of chemical libraries of small molecules for SLIT2/ROBO1 inhibition. Screening a focused chemical library of protein-protein interaction (PPI) inhibitors identified SMIFH2 as a SLIT2/ROBO1 inhibitor, demonstrating its ability to disrupt the interaction in a dose-dependent manner. Our study introduces a novel screening platform for identifying small molecule inhibitors of SLIT2/ROBO1, laying the foundation for future drug discovery efforts aimed at targeting this signaling axis in cancer and other diseases.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"34 ","pages":"Article 100240"},"PeriodicalIF":2.7,"publicationDate":"2025-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144113026","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 : 2025-05-05DOI: 10.1016/j.slasd.2025.100239
Arif Arrahman , Haifeng Xu , Muzaffar A. Khan , Tijmen S. Bos , Julien Slagboom , Guus C. van der Velden , Ulrike Nehrdich , Nicholas R. Casewell , Michael K. Richardson , Christian Tudorache , Fernanda C. Cardoso , Jeroen Kool
Snake venoms are complex bioactive mixtures designed to paralyse, kill, or digest prey. These venoms are of pharmacological interest due to their ability to modulate molecular targets such as ion channels and receptors with high specificity and potency. Traditional studies often focus on in vitro molecular analysis or in vivo behavioural effects, limiting comprehensive understanding. Here, we present a high-throughput screening platform that combines in vitro ion channel assays with in vivo zebrafish larval bioassays using nanofractionation analytics. This method integrates post-column calcium flux assays, zebrafish paralytic bioassays, toxin mass spectrometry, and proteomics to link bioactivity with toxin identification. Using elapid snake venoms (genus Dendroaspis, Naja, and Hemachatus) as a proof of concept, we identified several toxins modulating ion channels with paralytic effects on zebrafish larvae. Our approach enables parallel acquisition of in vitro and in vivo data, offering a robust guide for identifying and characterising ion channel modulators with defined molecular targets.
{"title":"Parallel in vitro ion channel and in vivo zebrafish assaying of elapid snake venoms following chromatographic separation of toxin components","authors":"Arif Arrahman , Haifeng Xu , Muzaffar A. Khan , Tijmen S. Bos , Julien Slagboom , Guus C. van der Velden , Ulrike Nehrdich , Nicholas R. Casewell , Michael K. Richardson , Christian Tudorache , Fernanda C. Cardoso , Jeroen Kool","doi":"10.1016/j.slasd.2025.100239","DOIUrl":"10.1016/j.slasd.2025.100239","url":null,"abstract":"<div><div>Snake venoms are complex bioactive mixtures designed to paralyse, kill, or digest prey. These venoms are of pharmacological interest due to their ability to modulate molecular targets such as ion channels and receptors with high specificity and potency. Traditional studies often focus on <em>in vitro</em> molecular analysis or <em>in vivo</em> behavioural effects, limiting comprehensive understanding. Here, we present a high-throughput screening platform that combines <em>in vitro</em> ion channel assays with <em>in vivo</em> zebrafish larval bioassays using nanofractionation analytics. This method integrates post-column calcium flux assays, zebrafish paralytic bioassays, toxin mass spectrometry, and proteomics to link bioactivity with toxin identification. Using elapid snake venoms (genus <em>Dendroaspis, Naja</em>, and <em>Hemachatus</em>) as a proof of concept, we identified several toxins modulating ion channels with paralytic effects on zebrafish larvae. Our approach enables parallel acquisition of <em>in vitro</em> and <em>in vivo</em> data, offering a robust guide for identifying and characterising ion channel modulators with defined molecular targets.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"34 ","pages":"Article 100239"},"PeriodicalIF":2.7,"publicationDate":"2025-05-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144026241","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 : 2025-05-03DOI: 10.1016/j.slasd.2025.100235
Hsiao-Tien Hagar , Virneliz Fernandez-Vega , Kuang-Wei Wang , Luis M. Ortiz Jordan , Justin Shumate , Louis Scampavia , April Sweet Tapayan , Hien M Nguyen , Timothy P. Spicer , Min-Hao Kuo
Alzheimer’s disease (AD) is a neurodegenerative disorder that affects more than 30 million people worldwide. Underlying the progressive decline of cognitive functions are the neurofibrillary tangles (NFTs) in neurons of the brain. The spatiotemporal distribution of NFTs predicts the progression of cognitive symptoms. In contrast, the senile plaques of amyloid-β aggregates, another major biomarker for AD, do not correlate with the clinical symptom development, consistent with the negligible benefits to cognitive functions in patients receiving anti-Aβ immunotherapies. A new drug discovery avenue targeting tau pathologies is therefore urgently needed. Using a recombinant hyperphosphorylated tau (p‐tau) that presents characters key to the disease, e.g., formation of neurotoxic aggregates, we conducted a fluorescence p-tau aggregation assay and completed a 100K-compound high-throughput screen (HTS) and identified inhibitors of p-tau aggregation and cytotoxicity. This dual functional screen resulted in several potent compounds that effectively curbed both p-tau aggregation and cytotoxicity. Results presented in this work are the first HTS for small-molecule compounds that target the cellular toxicity of hyperphosphorylated tau. Top hits found in this screen and their analogues to be developed in the near future may lead to breakthroughs in the therapeutic development for Alzheimer’s disease and other neurodegenerative tauopathies.
{"title":"Hyperphosphorylated tau-based Alzheimer’s Disease drug discovery: Identification of inhibitors of tau aggregation and cytotoxicity","authors":"Hsiao-Tien Hagar , Virneliz Fernandez-Vega , Kuang-Wei Wang , Luis M. Ortiz Jordan , Justin Shumate , Louis Scampavia , April Sweet Tapayan , Hien M Nguyen , Timothy P. Spicer , Min-Hao Kuo","doi":"10.1016/j.slasd.2025.100235","DOIUrl":"10.1016/j.slasd.2025.100235","url":null,"abstract":"<div><div>Alzheimer’s disease (AD) is a neurodegenerative disorder that affects more than 30 million people worldwide. Underlying the progressive decline of cognitive functions are the neurofibrillary tangles (NFTs) in neurons of the brain. The spatiotemporal distribution of NFTs predicts the progression of cognitive symptoms. In contrast, the senile plaques of amyloid-β aggregates, another major biomarker for AD, do not correlate with the clinical symptom development, consistent with the negligible benefits to cognitive functions in patients receiving anti-Aβ immunotherapies. A new drug discovery avenue targeting tau pathologies is therefore urgently needed. Using a recombinant hyperphosphorylated tau (p‐tau) that presents characters key to the disease, e.g., formation of neurotoxic aggregates, we conducted a fluorescence p-tau aggregation assay and completed a 100K-compound high-throughput screen (HTS) and identified inhibitors of p-tau aggregation and cytotoxicity. This dual functional screen resulted in several potent compounds that effectively curbed both p-tau aggregation and cytotoxicity. Results presented in this work are the first HTS for small-molecule compounds that target the cellular toxicity of hyperphosphorylated tau. Top hits found in this screen and their analogues to be developed in the near future may lead to breakthroughs in the therapeutic development for Alzheimer’s disease and other neurodegenerative tauopathies.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"33 ","pages":"Article 100235"},"PeriodicalIF":2.7,"publicationDate":"2025-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143902435","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 : 2025-05-01DOI: 10.1016/j.slasd.2025.100238
Robert K. Harmel , Christian N. Parker
{"title":"1st EU-OPENSCREEN/SLAS data mining competition to predict compounds solubility","authors":"Robert K. Harmel , Christian N. Parker","doi":"10.1016/j.slasd.2025.100238","DOIUrl":"10.1016/j.slasd.2025.100238","url":null,"abstract":"","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"34 ","pages":"Article 100238"},"PeriodicalIF":2.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144060291","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 : 2025-05-01DOI: 10.1016/j.slasd.2025.100237
Rukayat Aromokeye , Martha Ackerman-Berrier , Rosa del Carmen Araujo , Maria Lambousis , Savio Cardoza , L. Charlie Chen , Matthew E. Kaplan , Haining Zhu , Celina Zerbinatti , Christopher Penton , Gregory R.J. Thatcher , Timothy Marlowe
Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase and scaffolding protein that is primarily regulated by integrin signaling. FAK signaling increases cell motility in both normal and cancer cells, and FAK is often overexpressed and/or dysregulated in many types of cancer. FAK has three different domains: an N-terminal FERM domain, a central kinase domain (the traditional target for drug discovery), and a C-terminal focal adhesion targeting (FAT) domain. The FAT domain represents an alternative approach to targeting FAK, and our aim is to identify novel small molecules that will inhibit FAT protein-protein interactions (PPI), which may have implications for cancer and fibrosis treatment. Here, we describe the development and validation of a robust high-throughput screening (HTS) assay suitable for identifying inhibitors of the FAT:paxillin PPI. The 384-well low volume assay is based on time-resolved fluorescence resonance energy transfer (TR-FRET) technology and uses the high affinity biotin-PEG-1907 stapled peptide to mimic paxillin. We also present the development of a TR-FRET counterscreen assay using CD47 and SIRPα to detect nonspecific inhibitors, as well as an orthogonal surface plasmon resonance (SPR) binding assay. We employed the FAT: biotin-PEG-1907 assay to screen a 31,636-compound small molecule library. Primary positives (hits) from HTS were confirmed in concentration-response primary and counterscreen assays and validated in the SPR binding assay. We discovered 4 inhibitors of the FAT:paxillin PPI using this approach and established a framework for small molecule drug discovery efforts targeting the FAT domain of FAK.
{"title":"Development of a high-throughput TR-FRET assay to identify inhibitors of the FAK-paxillin protein-protein interaction","authors":"Rukayat Aromokeye , Martha Ackerman-Berrier , Rosa del Carmen Araujo , Maria Lambousis , Savio Cardoza , L. Charlie Chen , Matthew E. Kaplan , Haining Zhu , Celina Zerbinatti , Christopher Penton , Gregory R.J. Thatcher , Timothy Marlowe","doi":"10.1016/j.slasd.2025.100237","DOIUrl":"10.1016/j.slasd.2025.100237","url":null,"abstract":"<div><div>Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase and scaffolding protein that is primarily regulated by integrin signaling. FAK signaling increases cell motility in both normal and cancer cells, and FAK is often overexpressed and/or dysregulated in many types of cancer. FAK has three different domains: an N-terminal FERM domain, a central kinase domain (the traditional target for drug discovery), and a C-terminal focal adhesion targeting (FAT) domain. The FAT domain represents an alternative approach to targeting FAK, and our aim is to identify novel small molecules that will inhibit FAT protein-protein interactions (PPI), which may have implications for cancer and fibrosis treatment. Here, we describe the development and validation of a robust high-throughput screening (HTS) assay suitable for identifying inhibitors of the FAT:paxillin PPI. The 384-well low volume assay is based on time-resolved fluorescence resonance energy transfer (TR-FRET) technology and uses the high affinity biotin-PEG-1907 stapled peptide to mimic paxillin. We also present the development of a TR-FRET counterscreen assay using CD47 and SIRPα to detect nonspecific inhibitors, as well as an orthogonal surface plasmon resonance (SPR) binding assay. We employed the FAT: biotin-PEG-1907 assay to screen a 31,636-compound small molecule library. Primary positives (hits) from HTS were confirmed in concentration-response primary and counterscreen assays and validated in the SPR binding assay. We discovered 4 inhibitors of the FAT:paxillin PPI using this approach and established a framework for small molecule drug discovery efforts targeting the FAT domain of FAK.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"34 ","pages":"Article 100237"},"PeriodicalIF":2.7,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143927492","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 : 2025-04-28DOI: 10.1016/j.slasd.2025.100236
Laura J. Hsieh , Tracy Lou , Muryam A. Gourdet , Emily Wong , Geeta J. Narlikar
Chromatin states define cell fates and consequently dysfunctional chromatin states drive disease. Conventional approaches to target dysfunctional chromatin states typically rely on targeting a defined, structured binding pocket of a specific chromatin protein. However, drugs developed from targeting single chromatin proteins have often failed in the clinic due to toxicity from broad non-specific effects on the genome. Substantial previous work has indicated that the function of a given chromatin state is encoded in the context-dependent protein-protein interactions (PPIs) between the Intrinsically disordered regions (IDRs) and folded domains of the multiple constituents. Currently, there are no drug discovery approaches that target the complex multivalent protein interactions within a given dysfunctional chromatin state. Therefore, new methods are required to target chromatin within specific conformational contexts for better translation into humans. Prior discoveries from our group and others have shown that chromatin intrinsically forms condensates through weak, yet specific, multivalent interactions between itself and other components. Using this intrinsic property of chromatin, we have developed a new screening method to address this technology gap and identify modulators of dysfunctional chromatin states for drug discovery. Here, we show that we can recreate different chromatin contexts as phase-separated condensates that have distinct biochemical and biophysical properties. Furthermore, we have scaled the technology into a screening platform and identify small molecules that modulate chromatin states specifically based on their chromatin context. We anticipate that such specific targeting of a disease driving chromatin assembly would reduce off-target effects, translate better into humans and open a new landscape of therapeutic possibilities for targeting complex, multivalent interactions.
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