SLAS Discovery最新文献

{"title":"Discovery and validation of small molecule stabilizers of mutant triose phosphate isomerase (TPI) as potential lead candidates for TPI deficiency","authors":"Laura L. Vollmer ,&nbsp;Presley Roberts ,&nbsp;Samantha L. Eicher ,&nbsp;Marta Wołosowicz ,&nbsp;Priyal Patel ,&nbsp;Joseph R. Figura ,&nbsp;Ella R. Donahue ,&nbsp;Josh Berkowitz ,&nbsp;Dillon Gavlock ,&nbsp;Peter Wipf ,&nbsp;Matt LaPorte ,&nbsp;Steven J. Mullett ,&nbsp;Amelle Shillington ,&nbsp;Gregg E. Homanics ,&nbsp;Michael J. Palladino ,&nbsp;Andreas Vogt","doi":"10.1016/j.slasd.2025.100278","DOIUrl":"10.1016/j.slasd.2025.100278","url":null,"abstract":"<div><div><em>Triosephosphate Isomerase</em> deficiency (TPI-Df) is a devastating untreatable childhood metabolic disease resulting in anemia, severe locomotor impairment, and premature death. Numerous single amino acid substitutions in <em>TPI</em> are pathogenic and result in rapidly progressing multisystem disease. Importantly, all known pathogenic TPI-Df mutations result in a protein that retains function, and pathogenesis is known to result from decreased steady state levels of the functioning protein. There are no small molecule therapies for TPI-Df; current treatments are limited to symptomatic support and dietary interventions. We reasoned that a phenotypic screen was most appropriate to capture agents that stabilize mutant TPI and developed a human cellular TPI-Df assay based on a cellular model of the “common” TPI^E105D mutant protein fused with a GFP and a fluorescent ROS biosensor. The assay was implemented for high-content, high-throughput imaging, optimized to full HTS standards, and used to screen a 2,560 compound pilot library and the 220,700 compound NIH MLSMR compound collection to identify candidate compounds for development into small molecule TPI-Df therapies. Hits were validated in dose-response, TPI-Df patient cells, and various orthogonal assays. Limited SAR revealed three promising compound series, which were evaluated for potential mechanisms of action. The lead series had previously been identified as inducers of HIF1 alpha, spawning a novel hypothesis that HIF1 alpha activation might be a potential avenue to treat TPI-Df patients. A lead molecule was moved into preliminary mouse studies to evaluate pharmacokinetics and tissue distribution and was shown to be moderately brain-penetrant. The lead compound is now positioned for target identification studies and efficacy testing in vivo TPI Df models, including a newly validated mouse model.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"36 ","pages":"Article 100278"},"PeriodicalIF":2.7,"publicationDate":"2025-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145088523","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}

{"title":"Integrating AUROC and SSMD for quality control in high-throughput screening assays","authors":"Xiaohua Douglas Zhang","doi":"10.1016/j.slasd.2025.100269","DOIUrl":"10.1016/j.slasd.2025.100269","url":null,"abstract":"<div><div>High-throughput screening (HTS) assays are pivotal in modern biomedical research, particularly in drug discovery and functional genomics. Ensuring the quality and reliability of HTS data is critical, especially when dealing with the small sample sizes that are typical in such assays. This study explores the integration of two powerful statistical metrics—Strictly Standardized Mean Difference (SSMD) and Area Under the Receiver Operating Characteristic Curve (AUROC)—for quality control (QC) in HTS. SSMD offers a standardized, interpretable measure of effect size, while AUROC provides a threshold-independent assessment of discriminative power. By establishing the theoretical and empirical relationships between AUROC and SSMD, we demonstrate how these metrics complement each other and enhance QC practices. We provide parametric, semi-parametric, and non-parametric estimation methods, and demonstrate the utility of the integrated framework in real HTS datasets. Our findings support the joint application of SSMD and AUROC as a robust and interpretable approach to improving QC in HTS, particularly under constraints of limited sample sizes of positive and negative controls.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"36 ","pages":"Article 100269"},"PeriodicalIF":2.7,"publicationDate":"2025-09-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145006970","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}

{"title":"The role of m6A in lipid metabolism-related diseases","authors":"Qianhui Zeng ,&nbsp;Yuxuan Yang ,&nbsp;Yinquan Zhang ,&nbsp;Siwen Jiang","doi":"10.1016/j.slasd.2025.100257","DOIUrl":"10.1016/j.slasd.2025.100257","url":null,"abstract":"<div><div>N6-methyladenosine (m⁶A) modification represents one of the most abundant RNA epigenetic modifications in eukaryote organisms. This modification plays a crucial role in various RNA processing events through the actions of m⁶A methyltransferases, demethylases, and recognition proteins, thereby influencing a wide array of biological processes. Notably, the significance of m⁶A modification in lipid metabolism, along with its underlying molecular regulatory mechanisms, is increasingly being elucidated. The regulation of lipid metabolism is intricately linked to the maintenance of energy homeostasis, and disruptions in lipid metabolism are characteristic of numerous diseases, including obesity, non-alcoholic fatty liver disease, and cardiovascular disease. This review comprehensively summarizes the pivotal role and molecular mechanisms of m⁶A modification in diseases related to lipid metabolism, such as obesity, non-alcoholic fatty liver disease, and cardiovascular atherosclerosis. Additionally, it introduces pharmacological agents, plant extracts, and small molecule compounds that target m⁶A regulatory factors. This work provides theoretical references for the development of future therapeutic strategies targeting m⁶A modifications to treat diseases related to lipid metabolism.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100257"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144805449","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}

{"title":"Cytotoxic Profiling of Korea Chemical Bank Diversity Library","authors":"Jinhee Kim ,&nbsp;Kwang-Eun Choi ,&nbsp;Yuno Lee ,&nbsp;Daeyoung Jeong ,&nbsp;Hyun Young Kim ,&nbsp;Jung-In Lee ,&nbsp;Heeyeong Cho ,&nbsp;Nam-Chul Cho","doi":"10.1016/j.slasd.2025.100267","DOIUrl":"10.1016/j.slasd.2025.100267","url":null,"abstract":"<div><div>Cytotoxicity profiling of screening libraries is a critical component in early-stage drug discovery to identify compounds with undesirable toxic effects. Here, we report the cytotoxicity profiling of the Korea Chemical Bank (KCB) diversity library, comprising 7040 compounds curated via virtual screening, clustering, and druggability assessment. A subset of 5181 compounds was randomly selected and screened using the WST-1 assay in five mammalian cell lines (HEK293, HFL1, HepG2, NIH3T3, and CHOK1) at concentrations of 30 µM and 10 µM, following 24 h and 48 h incubation periods. Cytotoxic compounds were defined as those exhibiting &gt;50 % inhibition at 30 µM after 48 h. A total of 17 compounds showed consistent cytotoxicity across all five cell lines. Comparative analysis of physicochemical properties revealed that cytotoxic compounds exhibited higher lipophilicity (ALogP/ LogD) and the number of aromatic rings (AR) relative to non-cytotoxic compounds. These results indicate that the majority of the KCB diversity library comprises non-cytotoxic compounds, reflecting effective pre-filtering of toxic physicochemical properties during library design.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100267"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144921842","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}

{"title":"High-throughput evaluation of novel WRN inhibitors","authors":"Haiyan Xu ,&nbsp;Rachel L. Palte ,&nbsp;Meredith M. Rickard ,&nbsp;Soon Woo Kwon ,&nbsp;Xiaomei Chai ,&nbsp;Jing Yuan ,&nbsp;John Bassett ,&nbsp;Joseph Moran ,&nbsp;Markus Koglin ,&nbsp;Isaac Musisi ,&nbsp;Minjia Zhang ,&nbsp;Klaus Maskos ,&nbsp;Marcel J. Tauchert ,&nbsp;Yu-Shan Cheng ,&nbsp;Zixiong Wang ,&nbsp;Yi Yang ,&nbsp;Abhisek Banerjee ,&nbsp;Joanna L. Chen ,&nbsp;Indu Bharathan ,&nbsp;Lorena Rico ,&nbsp;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}

{"title":"Alternate dyes for image-based profiling assays","authors":"Suganya Sivagurunathan ,&nbsp;Patrick Byrne ,&nbsp;Alán F. Muñoz ,&nbsp;John Arevalo ,&nbsp;Anne E. Carpenter ,&nbsp;Shantanu Singh ,&nbsp;Maria Kost-Alimova ,&nbsp;Beth A. Cimini","doi":"10.1016/j.slasd.2025.100268","DOIUrl":"10.1016/j.slasd.2025.100268","url":null,"abstract":"<div><h3>Background</h3><div>Cell Painting, the leading image-based profiling assay, involves staining plated cells with six dyes that mark the different compartments in a cell. Such profiles can then be used to discover connections between samples (whether different cell lines, different genetic treatments, or different compound treatments) as well as to assess particular features impacted by each treatment. Researchers may wish to vary the standard dye panel to assess particular phenotypes, or image cells live while maintaining the ability to cluster profiles overall.</div></div><div><h3>Methods</h3><div>In this study, we evaluate the performance of dyes that can either replace or augment the traditional Cell Painting dyes or enable tracking live cell dynamics. We perturbed U2OS cells with 90 different compounds and subsequently stained them with either standard Cell Painting dyes (Revvity), or with MitoBrilliant (Tocris) replacing MitoTracker or Phenovue phalloidin 400LS (Revvity) replacing phalloidin. We also tested the live-cell compatible ChromaLive dye (Saguaro).</div></div><div><h3>Results</h3><div>All dye sets effectively separated biological replicates of the same sample vs. negative controls (phenotypic activity), although separating from replicates of all other compounds (phenotypic distinctiveness) proved challenging for all dye sets. While individual dye substitutions within the standard Cell Painting panel had minimal impact on assay performance, the live cell dye exhibited distinct performance profiles across different compound classes compared to the standard panel, with later time points more distinct than earlier ones.</div></div><div><h3>Discussion</h3><div>Substituting MitoBrilliant or Phenovue phalloidin 400LS for standard mitochondrial or actin dyes minimally impacted Cell Painting assay performance. Phenovue phalloidin 400LS offers the advantage of isolating actin features from Golgi or plasma membrane while accommodating an additional 568 nm dye. Live cell imaging, enabled by ChromaLive dye, provides real-time assessment of compound-induced morphological changes. Combining this with the standard Cell Painting assay significantly expands the feature space for enhanced cellular profiling. Our findings provide data-driven options for researchers selecting dye sets for image-based profiling.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"36 ","pages":"Article 100268"},"PeriodicalIF":2.7,"publicationDate":"2025-08-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144981862","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}

{"title":"Development of novel high-throughput biochemical competition assays to identify ligands of human asialoglycoprotein receptor 1","authors":"Jianming Liu ,&nbsp;Bradley Peter ,&nbsp;Lauren Rhodes ,&nbsp;Mats Ormö ,&nbsp;Bo Peng ,&nbsp;Pia Hansson ,&nbsp;Anders Gunnarsson ,&nbsp;Laurent Knerr ,&nbsp;Filip Miljković ,&nbsp;Maria Ölwegård-Halvarsson ,&nbsp;Mahya Dezfouli ,&nbsp;Niek Dekker ,&nbsp;Helena Lindmark ,&nbsp;Shalini Andersson","doi":"10.1016/j.slasd.2025.100265","DOIUrl":"10.1016/j.slasd.2025.100265","url":null,"abstract":"<div><div>Hepatocyte-specific Asialoglycoprotein receptor (ASGPR) and its native ligand N-acetylgalactosamine (GalNAc) have been actively exploited for targeted delivery of therapeutic and diagnostic agents to the liver. Identification of new potent ligands of ASGPR is of high interest to advance this field and expand to new applications in drug discovery. However, success of novel potent ASGPR ligand discovery has been limited due to the lack of robust high-throughput assays amenable to High-Throughput Screening (HTS). Here, we describe the design and development of two novel biochemical competition binding assays using recombinant human trimeric ASGR1 protein (ASGPR subunit 1) as a mimic of the native multimeric complex and a reference Alexa-647 fluorophore-labelled tri-GalNAc ligand as a tracer. Both ASGR1 TR-FRET and fluorescence polarization (FP) assays are in 384-well microplate format and have a large detection range (IC₅₀ of 2.5 nM - 100 µM), suitable for both monovalent and multivalent ASGPR ligands as well as oligonucleotide conjugates. The ASGR1 FP assay was miniaturized into a 1536-well assay format and a pilot screen of a small molecule library of about 7500 compounds was conducted, identifying 23 positive hits with IC₅₀ values between 12 - 100 µM. Five of the primary hits were validated in orthogonal TR-FRET and SPR binding assays and one of them was successfully docked into the ASGPR, with the docking pose closely matching the binding mode of structurally analogous compound found to be co-crystalized with ASGR1. The successful development of these new ASGR1 biochemical assays provides a new platform for an HTS campaign on small molecule collections to discover novel ASGPR ligands for liver-targeted delivery of efficient therapeutic agents, LYTACs or as potential drugs.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100265"},"PeriodicalIF":2.7,"publicationDate":"2025-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144917867","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}

{"title":"Orthogonal temperature-related intensity change (TRIC) and TR-FRET as a high-throughput screening platform for the discovery of SLIT2 binders: A proof-of-concept approach","authors":"Nelson García-Vázquez,&nbsp;Moustafa T. Gabr","doi":"10.1016/j.slasd.2025.100264","DOIUrl":"10.1016/j.slasd.2025.100264","url":null,"abstract":"<div><div>SLIT2, a secreted glycoprotein involved in axon guidance, immune modulation, and tumor progression, remains largely unexplored as a pharmacological target due to the absence of small-molecule modulators. Here, we present a proof-of-concept high-throughput screening platform that integrates Temperature-Related Intensity Change (TRIC) technology with time-resolved Förster resonance energy transfer (TR-FRET) to identify small molecules capable of disrupting the SLIT2/ROBO1 interaction. Screening a lipid metabolism–focused compound library (653 molecules) yielded bexarotene, as the most potent small molecule SLIT2 binder reported to date, with a dissociation constant (<em>K</em>_D) of 2.62 µM. Follow-up TR-FRET assays demonstrated dose-dependent inhibition of SLIT2/ROBO1 interaction, with relative half-maximal inhibitory concentration (relative IC₅₀) = 77.27 ± 17.32 µM, with a maximal inhibition (Imax) of ∼40 % at 400 µM. These findings suggest a novel extracellular activity of bexarotene and validate the combined use of TRIC and TR-FRET as a scalable screening strategy for SLIT2-targeted small molecules. This platform lays the groundwork for future high-throughput discovery efforts against SLIT2 and its signaling axis.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100264"},"PeriodicalIF":2.7,"publicationDate":"2025-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144863731","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}

{"title":"Cell based high-throughput screening for small molecule inhibitors of ATE1","authors":"Claudia McCown ,&nbsp;Evan A. Ambrose ,&nbsp;Devang M. Patel ,&nbsp;Hassan Al-Ali ,&nbsp;Louis Scampavia ,&nbsp;Fangliang Zhang ,&nbsp;Timothy P. Spicer","doi":"10.1016/j.slasd.2025.100263","DOIUrl":"10.1016/j.slasd.2025.100263","url":null,"abstract":"<div><div>Arginyltransferase 1 (ATE1) catalyzes post-translational arginylation, a process implicated in protein stability, cellular function, and disease pathology. Dysregulated arginylation is associated with neurodegenerative disorders, cancer, and inflammation. Particularly, the increase of ATE1 activity has been shown to cause cell death in response to acute stress, highlighting ATE1 as a promising therapeutic target. Despite its therapeutic relevance, no selective small-molecule inhibitors of ATE1 have been FDA-approved at this time, with previous screening efforts yielding compounds with high promiscuity and toxicity. This, in part, is due to the lack of assays that would accommodate large-scale screening for effective and safe ATE1-inhibitors. To address this challenge, we developed a cell-based high-throughput screening (HTS) assay utilizing a fluorescent reporter system based on an ATE1 substrate peptide fused to a fluorescence protein and co-expressed alongside another fluorescence protein for normalization. The assay enables real-time quantification of ATE1 activity by monitoring arginylation-dependent protein degradation within intact cells, measured by the ratio of the two fluorescence signals. We validated the assay in 96-well and 1536-well plate formats, demonstrating its scalability and robustness through key performance metrics, including Z'-factor and signal-to-background ratio. A pilot screen of a Library of Pharmacologically Active Compounds (LOPAC®1280) was performed to evaluate this approach. This study establishes a scalable and selective platform for discovering ATE1 inhibitors, paving the way for future therapeutic development targeting ATE1-mediated disease pathways.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100263"},"PeriodicalIF":2.7,"publicationDate":"2025-08-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144859975","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}

{"title":"High throughput screening for SARS-CoV-2 inhibitors targeting 5 helix bundle","authors":"Emery Smith ,&nbsp;Qibin Geng ,&nbsp;Justin Shumate ,&nbsp;Yuka Otsuka ,&nbsp;Louis Scampavia ,&nbsp;Thomas D. Bannister ,&nbsp;Timothy P. Spicer","doi":"10.1016/j.slasd.2025.100262","DOIUrl":"10.1016/j.slasd.2025.100262","url":null,"abstract":"<div><div>SARS-CoV-2 and other related viruses enter host cells via receptor recognition and membrane fusion. A crucial part of this is mediated by 5HB which is capable of binding to the viral spike heptad repeats (HR2) making 5HB a potential druggable target of virus entry. Thus, we constructed a 5-Helix Bundle (5HB) pentamer assay for the purpose of identifying potential inhibitors SARS-CoV-2 virus entry. Following implementation and optimization into a 1536 well format, we validated this assay via a pilot HTS and proved we were able to find small molecule inhibitors that appear to compete with the 5HB binding to HR2. This allowed us to push forward and complete the full HTS campaign testing 635,262 compounds. Upon completion of the 5HB pentamer HTS, we also tested and validated a monomer version of the 5HB assay against a pilot screen and then used it to help confirm on-target activity. This allowed for the selection of 130 compounds which were tested in dose titration format against the 5HB pentamer assay. The same compounds were tested in secondary cell-based assays for SARS2 and Machupo virus entry via a dual luciferase transient transfection system. We also incorporated a live/dead cytotoxicity counterscreen. At the conclusion of these screens, 41 compounds were found to be selective inhibitors of the 5HB pentamer assay. From these assays, 31 compounds and analogs were selected which were tested in both the pentamer and monomer assays. 5 compounds emerged which showed good potency in both assays which were then tested in the SARS pseudo virus assay to round out this exercise.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100262"},"PeriodicalIF":2.7,"publicationDate":"2025-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144840698","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}