SLAS Discovery最新文献

{"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}

{"title":"A high-throughput approach to evaluating NCp7 RNA binding activity for HIV-1 drug discovery","authors":"Joanna K. Winstone ,&nbsp;Rikki Uhrich ,&nbsp;Thibault Alle ,&nbsp;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":"2025-08-11","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}

{"title":"Identification of novel inflammasome inhibitors via cellular NLRP3 target engagement assays","authors":"Francisco Castillo ,&nbsp;Thomas A. Mackenzie ,&nbsp;Elisabeth Domingo ,&nbsp;Inmaculada Iañez ,&nbsp;Matthew B. Robers ,&nbsp;Jennifer Wilkinson ,&nbsp;Erika Kay-Tsumagari ,&nbsp;Martha O’Brien ,&nbsp;Olga Genilloud ,&nbsp;Rosario Fernandez-Godino ,&nbsp;Maria C. Ramos","doi":"10.1016/j.slasd.2025.100258","DOIUrl":"10.1016/j.slasd.2025.100258","url":null,"abstract":"<div><div>The NLRP3 (NOD-like receptor family, pyrin domain-containing protein 3) inflammasome, a multiprotein complex, plays a crucial role in triggering the release of pro-inflammatory cytokines like interleukin-1 beta. Abnormal activation of NLRP3 can mediate an aberrant immune response to viral infections and is associated with inflammatory diseases. In this study, the goal was to identify bioactive, potent, and specific inhibitors of NLRP3 that could modulate the inflammasome pathway and assess their potential therapeutic relevance. An innovative workflow was assembled by setting up a robust cellular-based high throughput screening (HTS) target engagement (TE) tool to identify potent NLRP3 inhibitors and validate their functional effect on the inflammasome downstream signaling cascade. A subset of 2,500 compounds from the European Chemical Biology Library (ECBL) was screened and validated inhibitors were subjected to a similarity study by state-of-the-art computational tools to comprehend their specific impact on inflammasomal signaling nodes upstream of NLRP3 and to propose feasible anti-inflammatory drugs. Ultimately, ten compounds were selected and validated in functional checkpoints of the NLRP3 inflammasome pathway, such as caspase-1 activity and IL-1β release, proving the validity of this HTS TE method for identifying NLRP3 inhibitors. Subsequent similarity-based clustering suggested the organization of the active compounds into three primary groups, linked to NF-κB signaling, ROS-induced NLRP3 activation, and NLRP3 induction pathway in response to microbial and related insults. Overall, these findings demonstrate the robustness and efficiency of the target-engagement methodology to capture bioactive inflammasome inhibitors with diverse mechanisms of action. Seven of the identified NLRP3 inhibitors were characterized as novel inflammasome inhibitors with therapeutic potential.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100258"},"PeriodicalIF":2.7,"publicationDate":"2025-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144823308","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":"An overview of drugging the bacterial cytoskeleton, rod, and divisome systems","authors":"Elvis Awuni","doi":"10.1016/j.slasd.2025.100261","DOIUrl":"10.1016/j.slasd.2025.100261","url":null,"abstract":"<div><div>Bacterial infections and antibiotic resistance remain significant threats to global health, with millions of related deaths recorded annually. Projections that antibacterial resistance-related deaths could reach alarming proportions in the coming years, along with the shortcomings of current interventions, highlight the need for new drug targets, novel antibiotics, and revised strategies and policy actions. The bacterial cytoskeleton, rod, and divisome systems (BCRDs) perform vital cellular roles and serve as a reserve of numerous potential therapeutic targets. The components of the BCRDs play different roles but share some relationships, suggesting the possibility of exploiting synergistic, polytherapeutic, and polypharmacological effects with antibiotics to mitigate bacterial resistance. Unfortunately, few drug targets within the BCRDs have been validated, and bacterial resistance to the inhibitors and approved antibiotics poses a challenge to the health and pharmaceutical industries. This review provides a concise but comprehensive overview of drugging the BCRDs, emphasizing the relationships and druggable potentials, validated targets, inhibitors, challenges, interventions, prospects, perspectives, and future directions geared toward reinvigorating research and overcoming bottlenecks in the sector. Overall, the material presented and discussed could facilitate the identification and validation of new therapeutic targets, the discovery and development of novel clinical drugs, and the revision of strategies and policy interventions to augment the fight against antibiotic resistance.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100261"},"PeriodicalIF":2.7,"publicationDate":"2025-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144818588","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":"Aurora 2.0: A fluorogenic dye library for expanding the capability of protein-adaptive differential scanning fluorimetry (paDSF)","authors":"Annemarie F. Charvat ,&nbsp;Kayleigh Mason-Chalmers ,&nbsp;Aneta Grabinska-Rogala ,&nbsp;Shloka Shivakumar ,&nbsp;Zachary Gale-Day ,&nbsp;Taiasean Wu ,&nbsp;Zoe Millbern ,&nbsp;Jonathan B. Grimm ,&nbsp;Emma C. Carroll ,&nbsp;K․ Peter R․ Nilsson ,&nbsp;Luke D. Lavis ,&nbsp;Nelson R. Vinueza ,&nbsp;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 (&lt; 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":"2025-08-08","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}