SLAS Discovery最新文献

{"title":"Rapid-response RNA-fluorescence in situ hybridization (FISH) assay platform for coronavirus antiviral high-throughput screening","authors":"Ryan Chan ,&nbsp;Christian Shema Mugisha ,&nbsp;Vorada Chuenchob ,&nbsp;Stephanie A. Moquin ,&nbsp;Ujjini H. Manjunatha ,&nbsp;Nadine Jarrousse ,&nbsp;Vineet D. Menachery ,&nbsp;Xuping Xie ,&nbsp;Erika L. Flannery ,&nbsp;Richard T. Eastman","doi":"10.1016/j.slasd.2024.100189","DOIUrl":"10.1016/j.slasd.2024.100189","url":null,"abstract":"<div><div>Over the past 25 years, the global community has faced challenges posed by three distinct outbreaks of coronaviruses including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The identification of a novel alphacoronavirus canine CoV (CCoV-HuPn2018) in human patients in Malaysia underscores the potential for crossover infections to humans. The threat of the ever-evolving nature of viral infections as well as the lingering health and socioeconomic effects of the recent SARS-CoV-2 pandemic emphasize the urgent need for advanced antiviral drug screening tools that can be quickly implemented to strengthen preparedness and preventive measures against future outbreaks. Here, we present the development and validation of a novel RNA-fluorescence <em>in situ</em> hybridization (FISH) imaging assay as a 384-well, high-throughput rapid response platform for antiviral drug discovery. RNA-FISH is a powerful tool to visualize specific mRNA in cultured cells using a high-content imaging platform. The flexibility of RNA-FISH probe sets allows for the rapid design of viral genome-specific probes, enabling <em>in vitro</em> assay development to test for inhibition of viral replication by either biologic or small molecule inhibitors. Screening of 170 antiviral compounds in concentration-response demonstrates a strong correlation between the RNA-FISH assay and an immunofluorescence assay (IFA) for both human coronaviruses HCoV-OC43 and HCoV-229E. Additionally, we successfully applied this methodology in the context of CCoV strain 1–71, proving rapid development and deployment, opening new avenues for the evaluation of antiviral drugs to potential future emerging threats.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-11-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142578690","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Magnetic 3D bioprinting of skeletal muscle spheroid for a spheroid-based screening assay","authors":"Chayanit Chaweewannakorn ,&nbsp;Khin The Nu Aye ,&nbsp;Joao N. Ferreira","doi":"10.1016/j.slasd.2024.100190","DOIUrl":"10.1016/j.slasd.2024.100190","url":null,"abstract":"<div><div>Over the past decade, there has been a rapid development in the use of magnetic three dimensional (3D) based cell culture systems. Concerning the skeletal muscle, 3D culture systems can provide biological insights for translational clinical research in the fields of muscle physiology and metabolism. These systems can enhance the cell culture environment by improving spatially-oriented cellular assemblies and morphological features closely mimicking the in vivo tissues/organs, since they promote strong interactions between cells and the extracellular matrix (ECM). However, the time-consuming and complex nature of 3D traditional culture techniques pose a challenge to the widespread adoption of 3D systems. Herein, a bench protocol is presented for creating an innovative, promptly assembled and user-friendly culture platform for the magnetic 3D bioprinting of skeletal muscle spheroids. Our protocol findings revealed consistent morphological outcomes and the functional development of skeletal muscle tissue, as evidenced by the expression of muscle-specific contractile proteins and myotubes and the responsiveness to stimulation with cholinergic neurotransmitters. This proof-of-concept protocol confirmed the future potential for further validation and application of spheroid-based assays in human skeletal muscle research.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142570581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Bioassay protocol metadata annotation: Proposed standards adoption","authors":"Rama Balakrishnan ,&nbsp;Ellen L. Berg ,&nbsp;Christopher C. Butler ,&nbsp;Alex M. Clark ,&nbsp;Sheryl P. Denker ,&nbsp;Isabella Feierberg ,&nbsp;Jason Harris ,&nbsp;Timothy P. Ikeda ,&nbsp;Samantha Jeschonek ,&nbsp;Vladimir A. Makarov ,&nbsp;Christopher Southan ,&nbsp;Dana Vanderwall ,&nbsp;Peter Winstanley","doi":"10.1016/j.slasd.2024.100188","DOIUrl":"10.1016/j.slasd.2024.100188","url":null,"abstract":"<div><div>We present a standardized metadata template for assays used in pharmaceutical drug discovery research, according to the FAIR principles. We also describe the use of an automated tool for annotating assays from a variety of sources, including PubChem, commercial assay providers, and the peer-reviewed literature, to this metadata template. Adoption of a standardized metadata template will allow drug discovery scientists to better understand and compare the increasing amounts of assay data becoming available, and will facilitate the use of artificial intelligence tools and other computational methods for analysis and prediction. Since bioassays drive advances in biomedical research, improvements in assay metadata can improve productivity in discovery of new therapeutics, platform technologies, and assay methods.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142482706","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A mechanism of action-reflective, dual cell-based bioassay for determining the bioactivity of sclerostin-neutralizing antibodies","authors":"Suzhen Wei ,&nbsp;Qiang Wu ,&nbsp;Chunlai Cao ,&nbsp;Zhuoni Yang ,&nbsp;Jianrui Shi ,&nbsp;Jingqun Huang ,&nbsp;Hua He ,&nbsp;Yongjie Lai ,&nbsp;Jing Li","doi":"10.1016/j.slasd.2024.100187","DOIUrl":"10.1016/j.slasd.2024.100187","url":null,"abstract":"<div><div>Osteoporosis is a major threat to the elderly worldwide. The Wnt signaling pathway plays a critical role in bone development and homeostasis. Sclerostin, a Wnt ligand inhibitor, competes with Wnt ligands for low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6) on osteoblasts, thereby suppressing bone formation. Sclerostin-neutralizing monoclonal antibodies (mAbs) have emerged as a potential bone-forming therapy for osteoporosis. A cell-based bioassay which determines the relative activity of a product, related to its mechanism of action, is of great importance from drug discovery to quality control and batch release. Currently used cell-based bioassays for sclerostin-neutralizing mAbs usually use Wnt1 or Wnt3a to stimulate the Wnt pathway; sclerostin is a direct inhibitor of Wnt1 but not Wnt3a. Wnt1 is a highly hydrophobic protein that binds to the producing cell membrane and acts in a juxtacrine manner to stimulate the Wnt pathway in neighboring cells. Bioassays for drugs that induce Wnt1 signaling should be performed in a juxtacrine manner. Here, we present a mechanism of action-reflective, dual cell-based reporter gene assay. In this assay, Wnt1 producer cells are co-cultured with cells containing the Wnt reporter genes, Wnt1 on the producer cells activates the Wnt signaling pathway in the reporter cells that are in direct cell-to-cell contact, and sclerostin-neutralizing mAbs specifically and effectively antagonize the sclerostin-mediated Wnt reporter gene suppression. This bioassay demonstrates good specificity, accuracy, linearity, and precision and is suitable for quality control, stability testing, batch release, and biosimilarity assessment of sclerostin-neutralizing mAbs.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142402302","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Recapitulation of NOD/RIPK2 signaling in iPSC-derived macrophages","authors":"Mozhgan Dehghan Harati ,&nbsp;Jim King ,&nbsp;Simon Langer ,&nbsp;Florian Binder ,&nbsp;Ralf Heilker","doi":"10.1016/j.slasd.2024.100185","DOIUrl":"10.1016/j.slasd.2024.100185","url":null,"abstract":"<div><div>Human induced pluripotent stem cell (iPSC)-derived macrophages (IDMs) present a valuable substitute for monocyte-derived macrophages (MDMs) in order to study inflammation pathways in vitro. Through optimization of an IDM differentiation protocol, a six-fold increase in the production yield of myeloid progenitors was achieved. The derived IDMs were further characterized with respect to nucleotide-binding oligomerization domain (NOD) and receptor-interacting serine/threonine-protein kinase 2 (RIPK2) signaling, a key regulatory pathway for autoimmune diseases. The IDM cells recapitulated MDM biology with respect to the proinflammatory chemokine and inflammatory cytokine fingerprint more closely than THP-1 cells. When assessing RIPK2 modulation effect on tumor necrosis factor α (TNF-α), a cardinal mediator of inflammation, a similar pharmacological effect of RIPK2 inhibitors was observed in IDMs and MDMs. Additionally, IDMs and MDMs displayed a similar transcription and pathway profile in response to NOD1/2 stimulation and pharmacological inhibition of RIPK2. In summary, the enhanced myeloid production yield in the improved IDM differentiation protocol offers new opportunities for utilizing physiologically relevant macrophage models in the context of inflammatory diseases.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142334048","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combination screen in multi-cell type tumor spheroids reveals interaction between aryl hydrocarbon receptor antagonists and E1 ubiquitin-activating enzyme inhibitor","authors":"Thomas S. Dexheimer ,&nbsp;Nathan P. Coussens ,&nbsp;Thomas Silvers ,&nbsp;Eric M. Jones ,&nbsp;Li Chen ,&nbsp;Jianwen Fang ,&nbsp;Joel Morris ,&nbsp;Jeffrey A. Moscow ,&nbsp;James H. Doroshow ,&nbsp;Beverly A. Teicher","doi":"10.1016/j.slasd.2024.100186","DOIUrl":"10.1016/j.slasd.2024.100186","url":null,"abstract":"<div><div>The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes of drug transporters and metabolic enzymes to detoxify small molecule xenobiotics. It has a complex role in cancer biology, influencing both the progression and suppression of tumors by modulating malignant properties of tumor cells and anti-tumor immunity, depending on the specific tumor type and developmental stage. This has led to the discovery and development of selective AhR modulators, including BAY 2416964 which is currently in clinical trials. To identify small molecule anticancer agents that might be combined with AhR antagonists for cancer therapy, a high-throughput combination screen was performed using multi-cell type tumor spheroids grown from malignant cells, endothelial cells, and mesenchymal stem cells. The AhR selective antagonists BAY 2416964, GNF351, and CH-223191 were tested individually and in combination with twenty-five small molecule anticancer agents. As single agents, BAY 2416964 and CH-223191 showed minimal activity, whereas GNF351 reduced the viability of some spheroid models at concentrations greater than 1 µM. The activity of most combinations aligned well with the single agent activity of the combined agent, without apparent contributions from the AhR antagonist. All three AhR antagonists sensitized tumor spheroids to TAK-243, an E1 ubiquitin-activating enzyme inhibitor. These combinations were active in spheroids containing bladder, breast, ovary, kidney, pancreas, colon, and lung tumor cell lines. The AhR antagonists also potentiated pevonedistat, a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit, in several tumor spheroid models. In contrast, the AhR antagonists did not enhance the cytotoxicity of the proteasome inhibitor bortezomib.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142373664","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Design considerations when creating a high throughput screen-compatible in vitro model of osteogenesis","authors":"Stephanie E. Doyle,&nbsp;Courtney N. Cazzola,&nbsp;Cynthia M. Coleman","doi":"10.1016/j.slasd.2024.100184","DOIUrl":"10.1016/j.slasd.2024.100184","url":null,"abstract":"<div><div>Inducing osteogenic differentiation <em>in vitro</em> is useful for the identification and development of bone regeneration therapies as well as modelling bone disorders. To couple <em>in vitro</em> models with high throughput screening techniques retains the assay's relevance in research while increasing its therapeutic impact. Miniaturizing, automating and/or digitalizing <em>in vitro</em> assays will reduce the required quantity of cells, biologic stimulants, culture/output assay reagents, time and cost. This review highlights the design and workflow considerations for creating a high throughput screen-compatible model of osteogenesis, comparing and contrasting osteogenic cell type, assay fabrication and culture methodology, osteogenic induction approach and repurposing existing output techniques.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-10-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142309323","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of an automated 3D high content cell screening platform for organoid phenotyping","authors":"Suleyman B. Bozal ,&nbsp;Greg Sjogren ,&nbsp;Antonio P. Costa ,&nbsp;Joseph S. Brown ,&nbsp;Shannon Roberts ,&nbsp;Dylan Baker ,&nbsp;Paul Gabriel Jr. ,&nbsp;Benjamin T. Ristau ,&nbsp;Michael Samuels ,&nbsp;William F. Flynn ,&nbsp;Paul Robson ,&nbsp;Elise T. Courtois","doi":"10.1016/j.slasd.2024.100182","DOIUrl":"10.1016/j.slasd.2024.100182","url":null,"abstract":"<div><p>The use of organoid models in biomedical research has grown substantially since their inception. As they gain popularity among scientists seeking more complex and biologically relevant systems, there is a direct need to expand and clarify potential uses of such systems in diverse experimental contexts. Herein we outline a high-content screening (HCS) platform that allows researchers to screen drugs or other compounds against three-dimensional (3D) cell culture systems in a multi-well format (384-well). Furthermore, we compare the quality of robotic liquid handling with manual pipetting and characterize and contrast the phenotypic effects detected by confocal imaging and biochemical assays in response to drug treatment. We show that robotic liquid handling is more consistent and amendable to high throughput experimental designs when compared to manual pipetting due to improved precision and automated randomization capabilities. We also show that image-based techniques are more sensitive to detecting phenotypic changes within organoid cultures than traditional biochemical assays that evaluate cell viability, supporting their integration into organoid screening workflows. Finally, we highlight the enhanced capabilities of confocal imaging in this organoid screening platform as they relate to discerning organoid drug responses in single-well co-cultures of organoids derived from primary human biopsies and patient-derived xenograft (PDX) models. Altogether, this platform enables automated, imaging-based HCS of 3D cellular models in a non-destructive manner, opening the path to complementary analysis through integrated downstream methods.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000443/pdfft?md5=95bb4a25e3d1035eb2e9758c7aafa3a7&pid=1-s2.0-S2472555224000443-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142156865","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High throughput screening for SARS-CoV-2 helicase inhibitors","authors":"Yuka Otsuka ,&nbsp;Eunjung Kim ,&nbsp;Austin Krueger ,&nbsp;Justin Shumate ,&nbsp;Chao Wang ,&nbsp;Bilel Bdiri ,&nbsp;Sultan Ullah ,&nbsp;HaJeung Park ,&nbsp;Louis Scampavia ,&nbsp;Thomas D. Bannister ,&nbsp;Donghoon Chung ,&nbsp;Timothy P. Spicer","doi":"10.1016/j.slasd.2024.100180","DOIUrl":"10.1016/j.slasd.2024.100180","url":null,"abstract":"<div><p>Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for nearly 7 million deaths worldwide since its outbreak in late 2019. Even with the rapid development and production of vaccines and intensive research, there is still a huge need for specific anti-viral drugs that address the rapidly arising new variants. To address this concern, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) Centers, tasked with exploring approaches to target pathogens with pandemic potential, including SARS-CoV-2. In this study, we sought inhibitors of SARS-CoV2 non-structural protein 13 (nsP13) as potential antivirals, first developing a HTS-compatible assay to measure SARS-CoV2 nsP13 helicase activity. Here we present our effort in implementing the assay in a 1,536 well-plate format and in identifying nsP13 inhibitor hit compounds from a ∼650,000 compound library. The primary screen was robust (average Z’ = 0.86 ± 0.05) and resulted in 7,009 primary hits. 1,763 of these compounds upon repeated retests were further confirmed, showing consistent inhibition. Following in-silico analysis, an additional orthogonal assay and titration assays, we identified 674 compounds with IC₅₀ &lt;10 μM. We confirmed activity of independent compound batches from de novo powders while also incorporating multiple counterscreen assays. Our study highlights the potential of this assay for use on HTS platforms to discover novel compounds inhibiting SARS-CoV2 nsP13, which merit further development as an effective SARS-CoV2 antiviral.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S247255522400042X/pdfft?md5=1a67cf248af10a04f27581fcaef8915e&pid=1-s2.0-S247255522400042X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142037948","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"SARS-CoV-2 Mpro inhibitor identification using a cellular gain-of-signal assay for high-throughput screening","authors":"Renee Delgado ,&nbsp;Jyoti Vishwakarma ,&nbsp;Seyed Arad Moghadasi ,&nbsp;Yuka Otsuka ,&nbsp;Justin Shumate ,&nbsp;Ashley Cuell ,&nbsp;Megan Tansiongco ,&nbsp;Christina B. Cooley ,&nbsp;Yanjun Chen ,&nbsp;Agnieszka Dabrowska ,&nbsp;Rahul Basu ,&nbsp;Paulina Duhita Anindita ,&nbsp;Dahai Luo ,&nbsp;Peter I. Dosa ,&nbsp;Daniel A. Harki ,&nbsp;Thomas Bannister ,&nbsp;Louis Scampavia ,&nbsp;Timothy P. Spicer ,&nbsp;Reuben S. Harris","doi":"10.1016/j.slasd.2024.100181","DOIUrl":"10.1016/j.slasd.2024.100181","url":null,"abstract":"<div><p>Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2, SARS2) is responsible for the COVID-19 pandemic and infections that continue to affect the lives of millions of people worldwide, especially those who are older and/or immunocompromised. The SARS2 main protease enzyme, M^pro (also called 3C-like protease, 3CL^pro), is a <em>bona fide</em> drug target as evidenced by potent inhibition with nirmatrelvir and ensitrelvir, the active components of the drugs Paxlovid and Xocova, respectively. However, the existence of nirmatrelvir and ensitrelvir-resistant isolates underscores the need to develop next-generation drugs with different resistance profiles and/or distinct mechanisms of action. Here, we report the results of a high-throughput screen of 649,568 compounds using a cellular gain-of-signal assay. In this assay, M^pro inhibits expression of a luciferase reporter, and 8,777 small molecules were considered hits by causing a gain in luciferase activity 3x SD above the sample field activity (6.8% gain-of-signal relative to 100 µM GC376). Single concentration and dose-response gain-of-signal experiments confirmed 3,522/8,762 compounds as candidate inhibitors. In parallel, all initial high-throughput screening hits were tested in a peptide cleavage assay with purified M^pro and only 39/8,762 showed inhibition. Importantly, 19/39 compounds (49%) re-tested positive in both SARS2 assays, including two previously reported M^pro inhibitors, demonstrating the efficacy of the overall screening strategy. This approach led to the rediscovery of known M^pro inhibitors such as calpain inhibitor II, as well as to the discovery of novel compounds that provide chemical information for future drug development efforts.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000431/pdfft?md5=9fdb1c3b7ba672d8f03e70671b9bc52f&pid=1-s2.0-S2472555224000431-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142037949","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}