Pub Date : 2025-06-01Epub 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.
{"title":"A biochemical screening platform to target chromatin states using condensates as a tool","authors":"Laura J. Hsieh , Tracy Lou , Muryam A. Gourdet , Emily Wong , Geeta J. Narlikar","doi":"10.1016/j.slasd.2025.100236","DOIUrl":"10.1016/j.slasd.2025.100236","url":null,"abstract":"<div><div>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.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"33 ","pages":"Article 100236"},"PeriodicalIF":2.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143902437","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 : 2025-06-01Epub 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":"2025-06-01","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 : 2025-06-01Epub Date: 2025-04-09DOI: 10.1016/j.slasd.2025.100232
Ghazala Muteeb , Raisa Nazir Ahmed Kazi , Mohammad Aatif , Asim Azhar , Mohamed El Oirdi , Mohd Farhan
Background
Antimicrobial resistance (AMR) develops into a worldwide health emergency through genetic and biochemical adaptations which enable microorganisms to resist antimicrobial treatment. β-lactamases (blaNDM, blaKPC) and efflux pumps (MexAB-OprM) working with mobile genetic elements facilitate fast proliferation of multidrug-resistant (MDR) and exttreme drug-resistant (XDR) phenotypes thus creating major concerns for healthcare systems and community health as well as the agricultural sector.
Objectives
The review dissimilarly unifies molecular resistance pathways with public health implications through the study of epidemiological data and monitoring approaches and innovative therapeutic solutions. Previous studies separating their attention between molecular genetics and clinical outcomes have been combined into our approach which delivers an all-encompassing analysis of AMR.
Key insights
The report investigates the resistance mechanisms which feature enzymatic degradation and efflux pump overexpression together with target modification and horizontal gene transfer because these factors represent important contributors to present-day AMR developments. This review investigates AMR effects on hospital and community environments where it affects pathogens including MRSA, carbapenem-resistant Klebsiella pneumoniae, and drug-resistant Pseudomonas aeruginosa. This document explores modern AMR management methods that comprise WHO GLASS molecular surveillance systems and three innovative strategies such as CRISPR-modified genome editing and bacteriophage treatments along with antimicrobial peptides and artificial intelligence diagnostic tools.
Conclusion
The resolution of AMR needs complete scientific and global operational methods alongside state-of-the-art therapeutic approaches. Worldwide management of drug-resistant infection burden requires both enhanced infection prevention procedures with next-generation antimicrobial strategies to reduce cases effectively.
{"title":"Antimicrobial resistance: Linking molecular mechanisms to public health impact","authors":"Ghazala Muteeb , Raisa Nazir Ahmed Kazi , Mohammad Aatif , Asim Azhar , Mohamed El Oirdi , Mohd Farhan","doi":"10.1016/j.slasd.2025.100232","DOIUrl":"10.1016/j.slasd.2025.100232","url":null,"abstract":"<div><h3>Background</h3><div>Antimicrobial resistance (AMR) develops into a worldwide health emergency through genetic and biochemical adaptations which enable microorganisms to resist antimicrobial treatment. β-lactamases (blaNDM, blaKPC) and efflux pumps (MexAB-OprM) working with mobile genetic elements facilitate fast proliferation of multidrug-resistant (MDR) and exttreme drug-resistant (XDR) phenotypes thus creating major concerns for healthcare systems and community health as well as the agricultural sector.</div></div><div><h3>Objectives</h3><div>The review dissimilarly unifies molecular resistance pathways with public health implications through the study of epidemiological data and monitoring approaches and innovative therapeutic solutions. Previous studies separating their attention between molecular genetics and clinical outcomes have been combined into our approach which delivers an all-encompassing analysis of AMR.</div></div><div><h3>Key insights</h3><div>The report investigates the resistance mechanisms which feature enzymatic degradation and efflux pump overexpression together with target modification and horizontal gene transfer because these factors represent important contributors to present-day AMR developments. This review investigates AMR effects on hospital and community environments where it affects pathogens including MRSA, carbapenem-resistant Klebsiella pneumoniae, and drug-resistant <em>Pseudomonas aeruginosa</em>. This document explores modern AMR management methods that comprise WHO GLASS molecular surveillance systems and three innovative strategies such as CRISPR-modified genome editing and bacteriophage treatments along with antimicrobial peptides and artificial intelligence diagnostic tools.</div></div><div><h3>Conclusion</h3><div>The resolution of AMR needs complete scientific and global operational methods alongside state-of-the-art therapeutic approaches. Worldwide management of drug-resistant infection burden requires both enhanced infection prevention procedures with next-generation antimicrobial strategies to reduce cases effectively.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"33 ","pages":"Article 100232"},"PeriodicalIF":2.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143843491","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 : 2025-04-01Epub Date: 2025-04-06DOI: 10.1016/j.slasd.2025.100228
Robert K. Harmel , Tanja Miletic , Katja Herzog , Bahne Stechmann
EU-OPENSCREEN is the European research infrastructure consortium for chemical biology and early drug discovery. It provides open access to high-throughput screening, chemoproteomics and spatial MS-based omics platforms and medicinal chemistry groups to support the discovery of new biologically active small molecules that act as starting points for the development of new chemical tool compounds and drugs. Since its inauguration in 2018, the research infrastructure evolved from a blueprint to a fully operational platform. As new trends and technologies have an important impact on modern drug discovery, EU-OPENSCREEN continuously expands and refines its portfolio of technologies and expertise. In this perspective, the key achievements of the past six years and the planned activities over the next years are described. We illustrate how scientists can benefit from EU-OPENSCREEN through gaining access to technology platforms and expertise to unlock the extraordinary potential of their research projects and translate them into novel, impactful and innovative applications.
{"title":"Empowering research in chemical biology and early drug discovery – an update from the European research infrastructure EU-OPENSCREEN","authors":"Robert K. Harmel , Tanja Miletic , Katja Herzog , Bahne Stechmann","doi":"10.1016/j.slasd.2025.100228","DOIUrl":"10.1016/j.slasd.2025.100228","url":null,"abstract":"<div><div>EU-OPENSCREEN is the European research infrastructure consortium for chemical biology and early drug discovery. It provides open access to high-throughput screening, chemoproteomics and spatial MS-based omics platforms and medicinal chemistry groups to support the discovery of new biologically active small molecules that act as starting points for the development of new chemical tool compounds and drugs. Since its inauguration in 2018, the research infrastructure evolved from a blueprint to a fully operational platform. As new trends and technologies have an important impact on modern drug discovery, EU-OPENSCREEN continuously expands and refines its portfolio of technologies and expertise. In this perspective, the key achievements of the past six years and the planned activities over the next years are described. We illustrate how scientists can benefit from EU-OPENSCREEN through gaining access to technology platforms and expertise to unlock the extraordinary potential of their research projects and translate them into novel, impactful and innovative applications.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100228"},"PeriodicalIF":2.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143812967","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 : 2025-04-01Epub Date: 2025-02-23DOI: 10.1016/j.slasd.2025.100222
Thomas S. Dexheimer , Zahra Davoudi , Nathan P. Coussens , Thomas Silvers , Joel Morris , Naoko Takebe , Rabih Said , Jeffrey A. Moscow , James H. Doroshow , Beverly A. Teicher
Dysregulation of the phosphatidylinositol 3-kinase (PI3K) pathway is a key contributor to cancer, making PI3K inhibitors a promising approach for targeted therapy. The selectivity of available inhibitors varies across different PI3K isoforms. Alpelisib and inavolisib are selective for the α-isoform, while duvelisib targets the δ- and γ-isoforms, and copanlisib is a pan-PI3K inhibitor, active against all isoforms. This study investigated the activity of these four PI3K inhibitors in combination with other targeted agents using multi-cell type tumor spheroids composed of 60% malignant cells, 25% endothelial cells, and 15% mesenchymal stem cells. Twenty-nine tumor spheroid models were evaluated, including twenty-six patient-derived cancer cell lines from the NCI Patient-Derived Models Repository and three established cell lines from the NCI-60 human tumor cell line panel. Additive and/or synergistic effects were observed with alpelisib or inavolisib or copanlisib in combination with a RAS/MEK/ERK pathway inhibitor, either selumetinib (MEK), ravoxertinib (ERK 1/2), or tovorafenib (DAY101, RAF). Combinations of each of these three PI3K inhibitors with the KRAS mutation specific inhibitors MTRX1133 (KRAS G12D) or sotorasib (KRAS G12C) had selective activity in cell lines harboring the corresponding target. Lastly, combination effects were observed from vertical inhibition of the PI3K/AKT/mTOR pathway with a PI3K inhibitor in combination with either the mTORC1/2 inhibitor sapanisertib or an AKT inhibitor, ipatasertib or afuresertib.
{"title":"Combinatorial screen of targeted agents with the PI3K inhibitors inavolisib, alpelisib, duvelisib, and copanlisib in multi-cell type tumor spheroids","authors":"Thomas S. Dexheimer , Zahra Davoudi , Nathan P. Coussens , Thomas Silvers , Joel Morris , Naoko Takebe , Rabih Said , Jeffrey A. Moscow , James H. Doroshow , Beverly A. Teicher","doi":"10.1016/j.slasd.2025.100222","DOIUrl":"10.1016/j.slasd.2025.100222","url":null,"abstract":"<div><div>Dysregulation of the phosphatidylinositol 3-kinase (PI3K) pathway is a key contributor to cancer, making PI3K inhibitors a promising approach for targeted therapy. The selectivity of available inhibitors varies across different PI3K isoforms. Alpelisib and inavolisib are selective for the α-isoform, while duvelisib targets the δ- and γ-isoforms, and copanlisib is a pan-PI3K inhibitor, active against all isoforms. This study investigated the activity of these four PI3K inhibitors in combination with other targeted agents using multi-cell type tumor spheroids composed of 60% malignant cells, 25% endothelial cells, and 15% mesenchymal stem cells. Twenty-nine tumor spheroid models were evaluated, including twenty-six patient-derived cancer cell lines from the NCI Patient-Derived Models Repository and three established cell lines from the NCI-60 human tumor cell line panel. Additive and/or synergistic effects were observed with alpelisib or inavolisib or copanlisib in combination with a RAS/MEK/ERK pathway inhibitor, either selumetinib (MEK), ravoxertinib (ERK 1/2), or tovorafenib (DAY101, RAF). Combinations of each of these three PI3K inhibitors with the KRAS mutation specific inhibitors MTRX1133 (KRAS G12D) or sotorasib (KRAS G12C) had selective activity in cell lines harboring the corresponding target. Lastly, combination effects were observed from vertical inhibition of the PI3K/AKT/mTOR pathway with a PI3K inhibitor in combination with either the mTORC1/2 inhibitor sapanisertib or an AKT inhibitor, ipatasertib or afuresertib.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100222"},"PeriodicalIF":2.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143506646","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 : 2025-04-01Epub Date: 2025-04-05DOI: 10.1016/j.slasd.2025.100229
Fengling Li , U Hang Chan , Julia Garcia Perez , Hong Zeng , Irene Chau , Yanjun Li , Almagul Seitova , Levon Halabelian
Human DExD/H-box RNA helicases are ubiquitous molecular motors that unwind and rearrange RNA secondary structures in an ATP-dependent manner. These enzymes play essential roles in nearly all aspects of RNA metabolism. While their biological functions are well-characterized, the kinetic mechanisms remain relatively understudied in vitro. In this study, we describe the development and optimization of a bioluminescence-based assay to characterize the ATPase activity of three human RNA helicases: MDA5, LGP2, and DDX1. The assays were conducted using annealed 24-mer ds-RNA (blunt-ended double-stranded RNA) or double-stranded RNA with a 25-nt 3ʹ overhang (partial ds-RNA). These findings establish a robust and high-throughput in vitro assay suitable for a 384-well format, enabling the discovery and characterization of inhibitors targeting MDA5, LGP2, and DDX1. This work provides a valuable resource for advancing our understanding of these helicases and their therapeutic potential in Alzheimer's disease.
{"title":"ATPase activity profiling of three human DExD/H-box RNA helicases","authors":"Fengling Li , U Hang Chan , Julia Garcia Perez , Hong Zeng , Irene Chau , Yanjun Li , Almagul Seitova , Levon Halabelian","doi":"10.1016/j.slasd.2025.100229","DOIUrl":"10.1016/j.slasd.2025.100229","url":null,"abstract":"<div><div>Human DExD/H-box RNA helicases are ubiquitous molecular motors that unwind and rearrange RNA secondary structures in an ATP-dependent manner. These enzymes play essential roles in nearly all aspects of RNA metabolism. While their biological functions are well-characterized, the kinetic mechanisms remain relatively understudied <em>in vitro</em>. In this study, we describe the development and optimization of a bioluminescence-based assay to characterize the ATPase activity of three human RNA helicases: MDA5, LGP2, and DDX1. The assays were conducted using annealed 24-mer ds-RNA (blunt-ended double-stranded RNA) or double-stranded RNA with a 25-nt 3ʹ overhang (partial ds-RNA). These findings establish a robust and high-throughput <em>in vitro</em> assay suitable for a 384-well format, enabling the discovery and characterization of inhibitors targeting MDA5, LGP2, and DDX1. This work provides a valuable resource for advancing our understanding of these helicases and their therapeutic potential in Alzheimer's disease.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100229"},"PeriodicalIF":2.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143805060","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 : 2025-04-01Epub Date: 2025-03-21DOI: 10.1016/j.slasd.2025.100227
Mya D. Gough , Matthew B. Robers , Cesear R. Corona , Ranjit K. Mehta , Mukesh K. Nyati , Peter L. Toogood
Pyruvate dehydrogenase kinases (PDHKs) are non-canonical serine/threonine kinases that regulate the pyruvate dehydrogenase complex. Given their central role in metabolism, dysregulation of PDHKs has been linked with a broad variety of pathological conditions, such as cardiovascular disease, diabetes, lactic acidosis, and cancer. While there are many small molecule PDHK inhibitors, including several that have advanced into clinical development, no PDHK inhibitor has been approved for therapeutic use for any indication. Currently the field lacks well-characterized tool compounds that can probe PDHK biology and differentiate between PDHK isoforms. Moreover, disconnects between biochemical and cell-based assays have complicated efforts to understand the biological effect of inhibiting PDHK catalytic activity. To better understand how PDHK inhibitors function in cells, we have developed a cell-based assay using NanoBRET Target Engagement technology. Here, we describe the use of NanoBRET to evaluate binding at the PDHK ATP and lipoamide sites. Using these assays, we have profiled previously described PDHK inhibitors and demonstrated the ability of NanoBRET to distinguish between PDHK inhibitors with different mechanisms of action and to elucidate isoform selectivity.
{"title":"Development of a cell-based target engagement assay for pyruvate dehydrogenase kinase","authors":"Mya D. Gough , Matthew B. Robers , Cesear R. Corona , Ranjit K. Mehta , Mukesh K. Nyati , Peter L. Toogood","doi":"10.1016/j.slasd.2025.100227","DOIUrl":"10.1016/j.slasd.2025.100227","url":null,"abstract":"<div><div>Pyruvate dehydrogenase kinases (PDHKs) are non-canonical serine/threonine kinases that regulate the pyruvate dehydrogenase complex. Given their central role in metabolism, dysregulation of PDHKs has been linked with a broad variety of pathological conditions, such as cardiovascular disease, diabetes, lactic acidosis, and cancer. While there are many small molecule PDHK inhibitors, including several that have advanced into clinical development, no PDHK inhibitor has been approved for therapeutic use for any indication. Currently the field lacks well-characterized tool compounds that can probe PDHK biology and differentiate between PDHK isoforms. Moreover, disconnects between biochemical and cell-based assays have complicated efforts to understand the biological effect of inhibiting PDHK catalytic activity. To better understand how PDHK inhibitors function in cells, we have developed a cell-based assay using NanoBRET Target Engagement technology. Here, we describe the use of NanoBRET to evaluate binding at the PDHK ATP and lipoamide sites. Using these assays, we have profiled previously described PDHK inhibitors and demonstrated the ability of NanoBRET to distinguish between PDHK inhibitors with different mechanisms of action and to elucidate isoform selectivity.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100227"},"PeriodicalIF":2.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143694675","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 : 2025-04-01Epub Date: 2025-04-11DOI: 10.1016/j.slasd.2025.100234
Dalibor Nakládal , Rick Oerlemans , Miroslava Molitorisová , Nikola Chomaničová , Gabriel Zorkócy , Christina Yoseif , Adrianus Cornelis van der Graaf , Stanislav Stuchlík , Guido Krenning , Matthew R. Groves , André Heeres , Zdenko Levarski , Ján Kyselovič , Rob H. Henning , Leo E. Deelman
Hyperhomocysteinemia is a risk factor for cardiovascular disease, neurological disorders, and bone abnormalities. The key enzyme in homocysteine metabolism, cystathionine-β-synthase (CBS) is recognized as a target for new homocysteine-lowering therapies including enzyme replacement and gene therapy. Currently, there are no pharmacotherapies available that enhance CBS activity through its allosteric mechanism. The only known allosteric activator of CBS is S-adenosyl-L-methionine (SAM), which is available as a food supplement, but its effectiveness is limited by low membrane permeability and universal involvement in methylation reactions as a substrate. The discovery of CBS activators in high-throughput screening is challenging due to a lack of dedicated assays. Available HTS-compatible activity assays for CBS rely on measuring the product hydrogen sulfide or methanethiol where the signal increases with increased CBS activity. In the case of fluorescence-based assays, it is challenging to discern activators from autofluorescent compounds.
In this study, we introduce a homocysteine consumption assay for isolated human CBS (HconCBS) based on the absorbance of Ellman's reagent. This assay leverages a decrease in signal upon CBS activation, with performance parameters exceeding the requirements for high-throughput screening. In a commercial library of 3010 compounds, the HconCBS assay identified 10 hit compounds as more active than SAM, whereas a fluorescence-based assay using 7-azido-4-methylcoumarin (AzMC) identified 141 hits. HconCBS identified 101 compounds with autoabsorbance which did not include hit compounds, while the fluorescence-based assay identified 383 autofluorescent compounds which included all hit compounds. While 4 out of 10 HconCBS hits were confirmed when purchased from a new source, the compounds affected homocysteine rather than CBS. Nevertheless, HconCBS consistently detected the CBS activator seleno-adenosyl-L-methionine (SeAM) added to 4 library plates and re-discovered the same library hits in 3 out of 4 re-screened plates.
Taken together, HconCBS was designed to enable the discovery of allosteric CBS activators with greater reliability than fluorescence-based methods. Despite identifying some compounds that acted on homocysteine rather than CBS, the assay consistently identified the CBS activators SAM and SeAM and demonstrated reproducibility across two screening rounds. These findings establish HconCBS as a valuable tool for identifying potential therapeutic candidates for hyperhomocysteinemia, addressing a key gap in the development of CBS-targeted pharmacotherapies.
{"title":"New homocysteine consumption assay for high-throughput screening of human cystathionine-β-synthase","authors":"Dalibor Nakládal , Rick Oerlemans , Miroslava Molitorisová , Nikola Chomaničová , Gabriel Zorkócy , Christina Yoseif , Adrianus Cornelis van der Graaf , Stanislav Stuchlík , Guido Krenning , Matthew R. Groves , André Heeres , Zdenko Levarski , Ján Kyselovič , Rob H. Henning , Leo E. Deelman","doi":"10.1016/j.slasd.2025.100234","DOIUrl":"10.1016/j.slasd.2025.100234","url":null,"abstract":"<div><div>Hyperhomocysteinemia is a risk factor for cardiovascular disease, neurological disorders, and bone abnormalities. The key enzyme in homocysteine metabolism, cystathionine-β-synthase (CBS) is recognized as a target for new homocysteine-lowering therapies including enzyme replacement and gene therapy. Currently, there are no pharmacotherapies available that enhance CBS activity through its allosteric mechanism. The only known allosteric activator of CBS is S-adenosyl-<span>L</span>-methionine (SAM), which is available as a food supplement, but its effectiveness is limited by low membrane permeability and universal involvement in methylation reactions as a substrate. The discovery of CBS activators in high-throughput screening is challenging due to a lack of dedicated assays. Available HTS-compatible activity assays for CBS rely on measuring the product hydrogen sulfide or methanethiol where the signal increases with increased CBS activity. In the case of fluorescence-based assays, it is challenging to discern activators from autofluorescent compounds.</div><div>In this study, we introduce a homocysteine consumption assay for isolated human CBS (HconCBS) based on the absorbance of Ellman's reagent. This assay leverages a decrease in signal upon CBS activation, with performance parameters exceeding the requirements for high-throughput screening. In a commercial library of 3010 compounds, the HconCBS assay identified 10 hit compounds as more active than SAM, whereas a fluorescence-based assay using 7-azido-4-methylcoumarin (AzMC) identified 141 hits. HconCBS identified 101 compounds with autoabsorbance which did not include hit compounds, while the fluorescence-based assay identified 383 autofluorescent compounds which included all hit compounds. While 4 out of 10 HconCBS hits were confirmed when purchased from a new source, the compounds affected homocysteine rather than CBS. Nevertheless, HconCBS consistently detected the CBS activator seleno-adenosyl-<span>L</span>-methionine (SeAM) added to 4 library plates and re-discovered the same library hits in 3 out of 4 re-screened plates.</div><div>Taken together, HconCBS was designed to enable the discovery of allosteric CBS activators with greater reliability than fluorescence-based methods. Despite identifying some compounds that acted on homocysteine rather than CBS, the assay consistently identified the CBS activators SAM and SeAM and demonstrated reproducibility across two screening rounds. These findings establish HconCBS as a valuable tool for identifying potential therapeutic candidates for hyperhomocysteinemia, addressing a key gap in the development of CBS-targeted pharmacotherapies.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100234"},"PeriodicalIF":2.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143834488","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 : 2025-04-01Epub Date: 2025-02-28DOI: 10.1016/j.slasd.2025.100223
Jenna Bradley , Patrick O'Shea , Catherine Wrench , Johann Mattsson , Roxane Paulin , Catherine Overed-Sayer , Laura Rosenberg , Henric Olsson , Davide Gianni
Senescent cells contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease with significant unmet need and therefore, there is an interest in discovering new drug targets that regulate this process. We design and perform a phenotypic screen with a secreted protein library in primary human lung fibroblasts to identify modulators of cell senescence. We identify FGF9 as a suppressor of several senescence phenotypes reducing stimulated p21 expression, enlarged morphology, DNA damage and SASP secretion, which is consistent with both DNA-damage and ROS induced senescence. We also show that FGF9 reduces fibroblast activation in both healthy and IPF fibroblasts shown by a reduction in pro-fibrotic markers such as α-smooth muscle actin and COL1A1 mRNA. Our findings identify FGF9 as a suppressor of both senescence and fibrotic features in lung fibroblasts and therefore could be targeted as a new therapeutic strategy for respiratory diseases such as IPF.
{"title":"A secretome screen in primary human lung fibroblasts identifies FGF9 as a novel regulator of cellular senescence","authors":"Jenna Bradley , Patrick O'Shea , Catherine Wrench , Johann Mattsson , Roxane Paulin , Catherine Overed-Sayer , Laura Rosenberg , Henric Olsson , Davide Gianni","doi":"10.1016/j.slasd.2025.100223","DOIUrl":"10.1016/j.slasd.2025.100223","url":null,"abstract":"<div><div>Senescent cells contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease with significant unmet need and therefore, there is an interest in discovering new drug targets that regulate this process. We design and perform a phenotypic screen with a secreted protein library in primary human lung fibroblasts to identify modulators of cell senescence. We identify FGF9 as a suppressor of several senescence phenotypes reducing stimulated p21 expression, enlarged morphology, DNA damage and SASP secretion, which is consistent with both DNA-damage and ROS induced senescence. We also show that FGF9 reduces fibroblast activation in both healthy and IPF fibroblasts shown by a reduction in pro-fibrotic markers such as α-smooth muscle actin and COL1A1 mRNA. Our findings identify FGF9 as a suppressor of both senescence and fibrotic features in lung fibroblasts and therefore could be targeted as a new therapeutic strategy for respiratory diseases such as IPF.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100223"},"PeriodicalIF":2.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143538194","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 : 2025-04-01Epub Date: 2025-02-11DOI: 10.1016/j.slasd.2025.100221
Nihal Bharath , Emma DiPietro , Olivia Durfee , Ina Kycia , Jennifer Splaine , Praveen Sethupathy , Michael S. Rogers , Khashayar Vakili
Fibrolamellar carcinoma (FLC) is a primary liver cancer with a poor prognosis, primarily due to the lack of effective chemotherapeutic options. The DNAJB1-PRKACA (DP) gene fusion is recognized as the key oncogenic driver in FLC. This fusion arises from a ∼400 kb heterozygous deletion on chromosome 19, which fuses exon 1 of DNAJB1 with exons 2–10 of PRKACA, the gene encoding the catalytic subunit of protein kinase A (PKA). While targeting DP is considered a promising therapeutic approach, attempts to inhibit the kinase function of the DP fusion protein have been largely unsuccessful due to off-target effects on wild-type PKA.
In response to this challenge, we developed a high-throughput screening (HTS) assay to identify inhibitors of DP's downstream signaling pathways involved in transcriptional regulation. Our previous research identified LINC00473 as a transcriptional marker for DP protein expression, and LINC00473 is known to be upregulated in FLC tumors. Additionally, evidence suggests that LINC00473 promotes FLC tumor growth.
Based on the relationship between DP and LINC00473 expression, we engineered the HEK-DP-Luc reporter cell line by modifying HEK293 cells to express DP at the endogenous locus and to express the NanoLuc luciferase gene under the control of the LINC00473 promoter and enhancer. We have optimized the HEK-DP-Luc cells for HTS, and here we present our pipeline for primary screening and counter-screening to identify compounds that inhibit DP's downstream transcriptional activity. This HTS platform provides a novel approach for therapeutic drug discovery in FLC.
{"title":"A novel high-throughput screening platform to identify inhibitors of DNAJB1-PRKACA-driven transcriptional activity in fibrolamellar carcinoma","authors":"Nihal Bharath , Emma DiPietro , Olivia Durfee , Ina Kycia , Jennifer Splaine , Praveen Sethupathy , Michael S. Rogers , Khashayar Vakili","doi":"10.1016/j.slasd.2025.100221","DOIUrl":"10.1016/j.slasd.2025.100221","url":null,"abstract":"<div><div>Fibrolamellar carcinoma (FLC) is a primary liver cancer with a poor prognosis, primarily due to the lack of effective chemotherapeutic options. The <em>DNAJB1-PRKACA</em> (DP) gene fusion is recognized as the key oncogenic driver in FLC. This fusion arises from a ∼400 kb heterozygous deletion on chromosome 19, which fuses exon 1 of <em>DNAJB1</em> with exons 2–10 of <em>PRKACA</em>, the gene encoding the catalytic subunit of protein kinase A (PKA). While targeting DP is considered a promising therapeutic approach, attempts to inhibit the kinase function of the DP fusion protein have been largely unsuccessful due to off-target effects on wild-type PKA.</div><div>In response to this challenge, we developed a high-throughput screening (HTS) assay to identify inhibitors of DP's downstream signaling pathways involved in transcriptional regulation. Our previous research identified <em>LINC00473</em> as a transcriptional marker for DP protein expression, and <em>LINC00473</em> is known to be upregulated in FLC tumors. Additionally, evidence suggests that <em>LINC00473</em> promotes FLC tumor growth.</div><div>Based on the relationship between DP and <em>LINC00473</em> expression, we engineered the HEK-DP-Luc reporter cell line by modifying HEK293 cells to express DP at the endogenous locus and to express the NanoLuc luciferase gene under the control of the <em>LINC00473</em> promoter and enhancer. We have optimized the HEK-DP-Luc cells for HTS, and here we present our pipeline for primary screening and counter-screening to identify compounds that inhibit DP's downstream transcriptional activity. This HTS platform provides a novel approach for therapeutic drug discovery in FLC.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"32 ","pages":"Article 100221"},"PeriodicalIF":2.7,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143416366","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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