Pub Date : 2025-09-01Epub Date: 2025-08-11DOI: 10.1016/j.slasd.2025.100260
Joanna K. Winstone , Rikki Uhrich , Thibault Alle , Brian C. Kraemer
The HIV-1 epidemic broadly impacts healthcare. There remains a continued need for improved anti-viral therapies resilient to the development of drug resistance. HIV-1 nucleocapsid protein 7 (NCp7) seems a prime drug target due to its unique nucleic acid chaperone activity required for multiple viral processes. NCp7 RNA binding activity has been shown to increase viral production and infectivity within the host. Here we introduce a high-throughput AlphaScreen assay to evaluate NCp7 RNA binding activity and validate its specificity and sensitivity using a known inhibitor. We also demonstrate the utility of this assay by performing a drug-repurposing screen, which identified seven confirmed inhibitors of NCp7 RNA binding and two confirmed enhancers of NCp7 RNA binding. This tool will aid in future NCp7-targeted drug discovery initiatives for the treatment of HIV-1 infection.
{"title":"A high-throughput approach to evaluating NCp7 RNA binding activity for HIV-1 drug discovery","authors":"Joanna K. Winstone , Rikki Uhrich , Thibault Alle , Brian C. Kraemer","doi":"10.1016/j.slasd.2025.100260","DOIUrl":"10.1016/j.slasd.2025.100260","url":null,"abstract":"<div><div>The HIV-1 epidemic broadly impacts healthcare. There remains a continued need for improved anti-viral therapies resilient to the development of drug resistance. HIV-1 nucleocapsid protein 7 (NCp7) seems a prime drug target due to its unique nucleic acid chaperone activity required for multiple viral processes. NCp7 RNA binding activity has been shown to increase viral production and infectivity within the host. Here we introduce a high-throughput AlphaScreen assay to evaluate NCp7 RNA binding activity and validate its specificity and sensitivity using a known inhibitor. We also demonstrate the utility of this assay by performing a drug-repurposing screen, which identified seven confirmed inhibitors of NCp7 RNA binding and two confirmed enhancers of NCp7 RNA binding. This tool will aid in future NCp7-targeted drug discovery initiatives for the treatment of HIV-1 infection.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100260"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144850062","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-08-25DOI: 10.1016/j.slasd.2025.100266
Haiyan Xu , Rachel L. Palte , Meredith M. Rickard , Soon Woo Kwon , Xiaomei Chai , Jing Yuan , John Bassett , Joseph Moran , Markus Koglin , Isaac Musisi , Minjia Zhang , Klaus Maskos , Marcel J. Tauchert , Yu-Shan Cheng , Zixiong Wang , Yi Yang , Abhisek Banerjee , Joanna L. Chen , Indu Bharathan , Lorena Rico , Robert J. Bauer
DNA repair is a critical component for the maintenance of genomic stability and cancer prevention. Werner syndrome helicase (WRN), a RecQ family helicase involved in DNA double-strand break (DSB) repair, has been identified as a promising therapeutic target for multiple cancer types with high microsatellite instability (MSI-H). Microsatelite unstable tumors are characterized by a vulnerability in the DNA mismatch repair mechanism and depend on WRN for survival. Internal validation confirmed that CRISPR-mediated knockout of WRN was lethal in MSI-H, but not microsatellite stable (MSS) tumor cells. Additionally, this effect was confirmed as contingent upon the helicase activity of the enzyme. The challenge in targeting WRN lies in identifying inhibitors that effectively engage the helicase without causing toxicity to normal or microsatellite stable (MSS) cells. To address this challenge, we initiated a collaborative effort combining in vitro biochemical assays with cell-based assays using a panel of MSI and MSS cells. This approach aimed to evaluate compounds derived from knowledge-based designs as well as hits identified through our internal screening efforts, including cell-based phenotypic screens, Automated Ligand Identification System (ALIS), biochemical ADP glo HTS, and DEL. The assay suite comprises biochemical ATPase and helicase assays, in addition to cell viability and two target engagement assays. The primary functional target engagement assay utilized a high-content imaging method to detect a biomarker of DNA DSBs, using histone H2AX phosphorylation (pH2AX). A cellular thermal shift assay served as an orthogonal assessment of target engagement. This work enabled a knowledge-based drug discovery approach that leveraged structural design through computational modeling capabilities, resulting in a potent and novel series of spirocyclic WRN inhibitors specifically targeting MSI-H tumor cells. Our findings underscore the potential of WRN as a drug target for treating MSI-H cancers and emphasize the significance of interdisciplinary approaches in the discovery and advancement of new therapeutic agents.
{"title":"High-throughput evaluation of novel WRN inhibitors","authors":"Haiyan Xu , Rachel L. Palte , Meredith M. Rickard , Soon Woo Kwon , Xiaomei Chai , Jing Yuan , John Bassett , Joseph Moran , Markus Koglin , Isaac Musisi , Minjia Zhang , Klaus Maskos , Marcel J. Tauchert , Yu-Shan Cheng , Zixiong Wang , Yi Yang , Abhisek Banerjee , Joanna L. Chen , Indu Bharathan , Lorena Rico , Robert J. Bauer","doi":"10.1016/j.slasd.2025.100266","DOIUrl":"10.1016/j.slasd.2025.100266","url":null,"abstract":"<div><div>DNA repair is a critical component for the maintenance of genomic stability and cancer prevention. Werner syndrome helicase (WRN), a RecQ family helicase involved in DNA double-strand break (DSB) repair, has been identified as a promising therapeutic target for multiple cancer types with high microsatellite instability (MSI-H). Microsatelite unstable tumors are characterized by a vulnerability in the DNA mismatch repair mechanism and depend on WRN for survival. Internal validation confirmed that CRISPR-mediated knockout of WRN was lethal in MSI-H, but not microsatellite stable (MSS) tumor cells. Additionally, this effect was confirmed as contingent upon the helicase activity of the enzyme. The challenge in targeting WRN lies in identifying inhibitors that effectively engage the helicase without causing toxicity to normal or microsatellite stable (MSS) cells. To address this challenge, we initiated a collaborative effort combining in vitro biochemical assays with cell-based assays using a panel of MSI and MSS cells. This approach aimed to evaluate compounds derived from knowledge-based designs as well as hits identified through our internal screening efforts, including cell-based phenotypic screens, Automated Ligand Identification System (ALIS), biochemical ADP glo HTS, and DEL. The assay suite comprises biochemical ATPase and helicase assays, in addition to cell viability and two target engagement assays. The primary functional target engagement assay utilized a high-content imaging method to detect a biomarker of DNA DSBs, using histone H2AX phosphorylation (pH2AX). A cellular thermal shift assay served as an orthogonal assessment of target engagement. This work enabled a knowledge-based drug discovery approach that leveraged structural design through computational modeling capabilities, resulting in a potent and novel series of spirocyclic WRN inhibitors specifically targeting MSI-H tumor cells. Our findings underscore the potential of WRN as a drug target for treating MSI-H cancers and emphasize the significance of interdisciplinary approaches in the discovery and advancement of new therapeutic agents.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100266"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144921706","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-07-21DOI: 10.1016/j.slasd.2025.100253
Adam Zweifach
Statistical tests can be used to help determine whether experimental manipulations produce effects. In tests of means, when more than two groups are compared the total number of Type 1 errors (false positive results) increases unless a correction is used. Tukey’s method is thought to offer good control of the number of false positives and high statistical power when all pairwise comparisons are made. However, the number of replicates in laboratory experiments is often quite low, and small sample sizes can undermine assumptions underlying statistical methods. I used simulations to investigate how well ANOVA followed by different post-hoc tests controls the total number of false positives when there are 3- 6 experimental groups and 2- 6 experimental replicates, conditions that span the range of typical values. Tukey’s method, one of the most common, allows too many. I investigated 11 other approaches to controlling false positives and found that none is as effective as the simple Bonferroni correction or offers much more power. I conclude that researchers should not make all pairwise comparisons using ANOVA followed by Tukey’s method but instead use Bonferroni’s method on a limited number of pre-selected comparisons.
{"title":"Bonferroni’s method, not Tukey’s, should be used to control the total number of false positives when making multiple pairwise comparisons in experiments with few replicates","authors":"Adam Zweifach","doi":"10.1016/j.slasd.2025.100253","DOIUrl":"10.1016/j.slasd.2025.100253","url":null,"abstract":"<div><div>Statistical tests can be used to help determine whether experimental manipulations produce effects. In tests of means, when more than two groups are compared the total number of Type 1 errors (false positive results) increases unless a correction is used. Tukey’s method is thought to offer good control of the number of false positives and high statistical power when all pairwise comparisons are made. However, the number of replicates in laboratory experiments is often quite low, and small sample sizes can undermine assumptions underlying statistical methods. I used simulations to investigate how well ANOVA followed by different post-hoc tests controls the total number of false positives when there are 3- 6 experimental groups and 2- 6 experimental replicates, conditions that span the range of typical values. Tukey’s method, one of the most common, allows too many. I investigated 11 other approaches to controlling false positives and found that none is as effective as the simple Bonferroni correction or offers much more power. I conclude that researchers should not make all pairwise comparisons using ANOVA followed by Tukey’s method but instead use Bonferroni’s method on a limited number of pre-selected comparisons.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100253"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144700594","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-08-08DOI: 10.1016/j.slasd.2025.100259
Annemarie F. Charvat , Kayleigh Mason-Chalmers , Aneta Grabinska-Rogala , Shloka Shivakumar , Zachary Gale-Day , Taiasean Wu , Zoe Millbern , Jonathan B. Grimm , Emma C. Carroll , K․ Peter R․ Nilsson , Luke D. Lavis , Nelson R. Vinueza , Jason E. Gestwicki
Differential Scanning Fluorimetry (DSF) is a biophysical assay that is used to estimate protein stability in vitro. In a DSF experiment, the increased fluorescence of a solvatochromatic dye, such as Sypro Orange, is used to detect the unfolding of a protein during heating. However, Sypro Orange is only compatible with a minority of proteins (< 30 %), limiting the scope of this method. We recently reported that protein-adaptive DSF (paDSF) can partially solve this problem, wherein the protein is initially pre-screened against ∼300 chemically diverse dyes, termed the Aurora collection. While this approach significantly improves the number of targets amenable to DSF, it still fails to produce protein-dye pairs for some proteins. Here, we report the expansion of the dye collection to Aurora 2.0, which includes a total of 517 structurally diverse molecules and multiple new chemotypes. To assess performance, these dyes were screened against a panel of ∼100 proteins, which were selected, in part, to represent the most challenging targets (e.g. small size). From this effort, Aurora 2.0 achieved an impressive success rate of 94 %, including producing dyes for some targets that were not matched in the original collection. These findings support the idea that larger, more chemically diverse libraries improve the likelihood of detecting melting transitions across a wider range of proteins. We propose that Aurora 2.0 makes paDSF an increasingly powerful method for studying protein stability, ligand binding and other biophysical properties in high throughput.
{"title":"Aurora 2.0: A fluorogenic dye library for expanding the capability of protein-adaptive differential scanning fluorimetry (paDSF)","authors":"Annemarie F. Charvat , Kayleigh Mason-Chalmers , Aneta Grabinska-Rogala , Shloka Shivakumar , Zachary Gale-Day , Taiasean Wu , Zoe Millbern , Jonathan B. Grimm , Emma C. Carroll , K․ Peter R․ Nilsson , Luke D. Lavis , Nelson R. Vinueza , Jason E. Gestwicki","doi":"10.1016/j.slasd.2025.100259","DOIUrl":"10.1016/j.slasd.2025.100259","url":null,"abstract":"<div><div>Differential Scanning Fluorimetry (DSF) is a biophysical assay that is used to estimate protein stability <em>in vitro</em>. In a DSF experiment, the increased fluorescence of a solvatochromatic dye, such as Sypro Orange, is used to detect the unfolding of a protein during heating. However, Sypro Orange is only compatible with a minority of proteins (< 30 %), limiting the scope of this method. We recently reported that protein-adaptive DSF (paDSF) can partially solve this problem, wherein the protein is initially pre-screened against ∼300 chemically diverse dyes, termed the Aurora collection. While this approach significantly improves the number of targets amenable to DSF, it still fails to produce protein-dye pairs for some proteins. Here, we report the expansion of the dye collection to Aurora 2.0, which includes a total of 517 structurally diverse molecules and multiple new chemotypes. To assess performance, these dyes were screened against a panel of ∼100 proteins, which were selected, in part, to represent the most challenging targets (<em>e.g.</em> small size). From this effort, Aurora 2.0 achieved an impressive success rate of 94 %, including producing dyes for some targets that were not matched in the original collection. These findings support the idea that larger, more chemically diverse libraries improve the likelihood of detecting melting transitions across a wider range of proteins. We propose that Aurora 2.0 makes paDSF an increasingly powerful method for studying protein stability, ligand binding and other biophysical properties in high throughput.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100259"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144818589","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 : 2025-07-01Epub 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":"2025-07-01","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 : 2025-07-01Epub 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":"2025-07-01","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 : 2025-07-01Epub 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":"2025-07-01","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 : 2025-07-01Epub 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":"2025-07-01","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 : 2025-06-01Epub Date: 2025-04-09DOI: 10.1016/j.slasd.2025.100230
Nathan P. Coussens , Thomas S. Dexheimer , Thomas Silvers , Phillip R. Sanchez , Li Chen , Melinda G. Hollingshead , Naoko Takebe , James H. Doroshow , Beverly A. Teicher
Apoptosis, or programmed cell death, plays a critical role in maintaining tissue homeostasis by eliminating damaged or abnormal cells. Dysregulation of apoptosis pathways is a hallmark of cancer, allowing malignant cells to evade cell death and proliferate uncontrollably. Targeting apoptosis pathways has emerged as a promising therapeutic strategy in cancer treatment, aiming to restore the balance between cell survival and death. The MDM2 inhibitor alrizomadlin, the Bcl-2/Bcl-xL inhibitor pelcitoclax, and the IAP family inhibitor dasminapant were evaluated both individually and in combinations with standard of care and investigational anticancer small molecules in a spheroid model of solid tumors. The multi-cell type tumor spheroids were grown from human endothelial cells and mesenchymal stem cells combined with human malignant cells that were either established or patient-derived cell lines from the NCI Patient-Derived Models Repository. The malignant cell lines were derived from a range of solid tumors including uterine carcinosarcoma, synovial sarcoma, rhabdomyosarcoma, soft tissue sarcoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor (MPNST), pancreas, ovary, colon, breast, and small cell lung cancer. Interactions were observed from combinations of the apoptosis pathway targeted agents. Additionally, interactions were observed from combinations of the apoptosis pathway targeted agents with other agents, including PARP inhibitors, the XPO1 inhibitor eltanexor, and the PI3K inhibitor copanlisib. Enhanced activity was also observed from combinations of the apoptosis pathway targeted agents with MAPK pathway targeted agents, including the MEK inhibitor cobimetinib as well as adagrasib and MRTX1133, which specifically target the KRAS G12C and G12D variants, respectively.
{"title":"Combinatorial screen with apoptosis pathway targeted agents alrizomadlin, pelcitoclax, and dasminapant in multi-cell type tumor spheroids","authors":"Nathan P. Coussens , Thomas S. Dexheimer , Thomas Silvers , Phillip R. Sanchez , Li Chen , Melinda G. Hollingshead , Naoko Takebe , James H. Doroshow , Beverly A. Teicher","doi":"10.1016/j.slasd.2025.100230","DOIUrl":"10.1016/j.slasd.2025.100230","url":null,"abstract":"<div><div>Apoptosis, or programmed cell death, plays a critical role in maintaining tissue homeostasis by eliminating damaged or abnormal cells. Dysregulation of apoptosis pathways is a hallmark of cancer, allowing malignant cells to evade cell death and proliferate uncontrollably. Targeting apoptosis pathways has emerged as a promising therapeutic strategy in cancer treatment, aiming to restore the balance between cell survival and death. The MDM2 inhibitor alrizomadlin, the Bcl-2/Bcl-xL inhibitor pelcitoclax, and the IAP family inhibitor dasminapant were evaluated both individually and in combinations with standard of care and investigational anticancer small molecules in a spheroid model of solid tumors. The multi-cell type tumor spheroids were grown from human endothelial cells and mesenchymal stem cells combined with human malignant cells that were either established or patient-derived cell lines from the NCI Patient-Derived Models Repository. The malignant cell lines were derived from a range of solid tumors including uterine carcinosarcoma, synovial sarcoma, rhabdomyosarcoma, soft tissue sarcoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor (MPNST), pancreas, ovary, colon, breast, and small cell lung cancer. Interactions were observed from combinations of the apoptosis pathway targeted agents. Additionally, interactions were observed from combinations of the apoptosis pathway targeted agents with other agents, including PARP inhibitors, the XPO1 inhibitor eltanexor, and the PI3K inhibitor copanlisib. Enhanced activity was also observed from combinations of the apoptosis pathway targeted agents with MAPK pathway targeted agents, including the MEK inhibitor cobimetinib as well as adagrasib and MRTX1133, which specifically target the KRAS G12C and G12D variants, respectively.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"33 ","pages":"Article 100230"},"PeriodicalIF":2.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143859651","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: 2025-04-12DOI: 10.1016/j.slasd.2025.100233
Rae M. Sammons , Soma Ghosh , Lacin Yapindi , Eun Jeong Cho , Faye M. Johnson , Kevin N. Dalby
TRIP13, a promising target for cancer therapy, has been identified as a key regulator of the mitotic checkpoint. Overexpression of TRIP13 is associated with poor clinical outcomes in various cancers. Inhibition of TRIP13 has the potential to address therapeutic challenges in cancer, particularly in therapy-resistant and Rb-deficient cancers. Despite the potential therapeutic benefits of TRIP13 inhibition, the development of TRIP13 inhibitors has been hindered by the lack of a robust high-throughput screening (HTS) assay.
We developed a luminescence-based biochemical assay for TRIP13 activity to address this challenge using the ADP-Glo detection system. This assay offers high sensitivity, low background signal, and ease of automation, making it ideal for HTS applications. A pilot screen of kinase-focused inhibitors library and a large-scale screen of 4000 additional compounds demonstrated the assay's robust performance with a z'-factor exceeding 0.85 and a signal-to-background (S/B) ratio near 6. From the 50 initial hits, rigorous validation identified anlotinib as the most potent TRIP13 inhibitor with an IC50 of 5 μM. A cellular thermal shift assay (CETSA) confirmed the direct binding of anlotinib to TRIP13, validating the potential of our biochemical assay for identifying novel TRIP13 inhibitors. Our study provides a valuable tool for discovering novel TRIP13 inhibitors and advances our understanding of the therapeutic potential of targeting TRIP13 in cancer.
{"title":"Advancing the development of TRIP13 inhibitors: A high-throughput screening approach","authors":"Rae M. Sammons , Soma Ghosh , Lacin Yapindi , Eun Jeong Cho , Faye M. Johnson , Kevin N. Dalby","doi":"10.1016/j.slasd.2025.100233","DOIUrl":"10.1016/j.slasd.2025.100233","url":null,"abstract":"<div><div>TRIP13, a promising target for cancer therapy, has been identified as a key regulator of the mitotic checkpoint. Overexpression of TRIP13 is associated with poor clinical outcomes in various cancers. Inhibition of TRIP13 has the potential to address therapeutic challenges in cancer, particularly in therapy-resistant and Rb-deficient cancers. Despite the potential therapeutic benefits of TRIP13 inhibition, the development of TRIP13 inhibitors has been hindered by the lack of a robust high-throughput screening (HTS) assay.</div><div>We developed a luminescence-based biochemical assay for TRIP13 activity to address this challenge using the ADP-Glo detection system. This assay offers high sensitivity, low background signal, and ease of automation, making it ideal for HTS applications. A pilot screen of kinase-focused inhibitors library and a large-scale screen of 4000 additional compounds demonstrated the assay's robust performance with a z'-factor exceeding 0.85 and a signal-to-background (S/B) ratio near 6. From the 50 initial hits, rigorous validation identified anlotinib as the most potent TRIP13 inhibitor with an IC<sub>50</sub> of 5 μM. A cellular thermal shift assay (CETSA) confirmed the direct binding of anlotinib to TRIP13, validating the potential of our biochemical assay for identifying novel TRIP13 inhibitors. Our study provides a valuable tool for discovering novel TRIP13 inhibitors and advances our understanding of the therapeutic potential of targeting TRIP13 in cancer.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"33 ","pages":"Article 100233"},"PeriodicalIF":2.7,"publicationDate":"2025-06-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143855802","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}
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