SLAS Discovery最新文献

{"title":"Development of a time-resolved fluorescence resonance energy transfer ultra-high throughput screening assay targeting SYK and FCER1G interaction","authors":"Yuhong Du ,&nbsp;Dongxue Wang ,&nbsp;Vittorio L. Katis ,&nbsp;Elizabeth L. Zoeller ,&nbsp;Min Qui ,&nbsp;Allan I. Levey ,&nbsp;Opher Gileadi ,&nbsp;Haian Fu ,&nbsp;the Emory-SAGE-SGC TREAT-AD Center","doi":"10.1016/j.slasd.2024.100177","DOIUrl":"10.1016/j.slasd.2024.100177","url":null,"abstract":"<div><p>The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format. The assay utilizes the His-SH2 domains of SYK, which are indirectly labeled with anti-His-terbium to serve as a TR-FRET donor and a FITC-conjugated phosphorylated ITAM domain peptide of FCER1G to serve as an acceptor. We have optimized the assay into a 384-well HTS format and further miniaturized the assay into a 1536-well uHTS format. Robust assay performance has been achieved with a Z’ factor &gt; 0.8 and signal-to-background (S/B) ratio &gt; 15. The utilization of this uHTS TR-FRET assay for compound screening has been validated by a pilot screening of 2,036 FDA-approved and bioactive compounds library. Several primary hits have been identified from the pilot uHTS. One compound, hematoxylin, was confirmed to disrupt the SYK/FECR1G interaction in an orthogonal protein–protein interaction assay. Thus, our optimized and miniaturized uHTS assay could be applied to future scaling up of a screening campaign to identify small molecule inhibitors targeting the SYK and FCER1G interaction.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-08-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S247255522400039X/pdfft?md5=7e901da804daafea39d23b46e08f7bdf&pid=1-s2.0-S247255522400039X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142001528","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":"Simultaneous screening for selective SARS-CoV-2, Lassa, and Machupo virus entry inhibitors","authors":"Yuka Otsuka ,&nbsp;Lizhou Zhang ,&nbsp;Huihui Mou ,&nbsp;Justin Shumate ,&nbsp;Claire E. Kitzmiller ,&nbsp;Louis Scampavia ,&nbsp;Thomas D. Bannister ,&nbsp;Michael Farzan ,&nbsp;Hyeryun Choe ,&nbsp;Timothy P. Spicer","doi":"10.1016/j.slasd.2024.100178","DOIUrl":"10.1016/j.slasd.2024.100178","url":null,"abstract":"<div><p>Emerging highly pathogenic viruses can pose profound impacts on global health, the economy, and society. To meet that challenge, the National Institute of Allergy and Infectious Diseases (NIAID) established nine Antiviral Drug Discovery (AViDD) centers for early-stage identification and validation of novel antiviral drug candidates against viruses with pandemic potential. As part of this initiative, we established paired entry assays that simultaneously screen for inhibitors specifically targeting SARS-CoV-2 (SARS2), Lassa virus (LASV) and Machupo virus (MACV) entry. To do so we employed a dual pseudotyped virus (PV) infection system allowing us to screen ∼650,000 compounds efficiently and cost-effectively. Adaptation of these paired assays into 1536 well-plate format for ultra-high throughput screening (uHTS) resulted in the largest screening ever conducted in our facility, with over 2.4 million wells completed. The paired infection system allowed us to detect two PV infections simultaneously: LASV + MACV, MACV + SARS2, and SARS2 + LASV. Each PV contains a different luciferase reporter gene which enabled us to measure the infection of each PV exclusively, albeit in the same well. Each PV was screened at least twice utilizing different reporters, which allowed us to select the inhibitors specific to a particular PV and to exclude those that hit off targets, including cellular components or the reporter proteins. All assays were robust with an average Z’ value ranging from 0.5 to 0.8. The primary screening of ∼650,000 compounds resulted in 1812, 1506, and 2586 unique hits for LASV, MACV, and SARS2, respectively. The confirmation screening narrowed this list further to 60, 40, and 90 compounds that are unique to LASV, MACV, and SARS2, respectively. Of these compounds, 8, 35, and 50 compounds showed IC₅₀ value &lt; 10 μM, some of which have much greater potency and excellent antiviral activity profiles specific to LASV, MACV, and SARS2, and none are cytotoxic. These selected compounds are currently being studied for their mechanism of action and to improve their specificity and potency through chemical modification.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000406/pdfft?md5=fc5c2087563791142a95d69edf0d38c3&pid=1-s2.0-S2472555224000406-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142006028","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 a sensitive high-throughput enzymatic assay capable of measuring sub-nanomolar inhibitors of SARS-CoV2 Mpro","authors":"Peter Kovar,&nbsp;Paul L Richardson,&nbsp;Alla Korepanova,&nbsp;Gustavo A Afanador,&nbsp;Vladimir Stojkovic,&nbsp;Tao Li,&nbsp;Michael R Schrimpf,&nbsp;Teresa I Ng,&nbsp;David A Degoey,&nbsp;Sujatha M Gopalakrishnan,&nbsp;Jun Chen","doi":"10.1016/j.slasd.2024.100179","DOIUrl":"10.1016/j.slasd.2024.100179","url":null,"abstract":"<div><p>The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC₅₀ values determined in this assay correlates well with EC₅₀ values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-08-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000418/pdfft?md5=b7b058077a624412d931d004dd0e213e&pid=1-s2.0-S2472555224000418-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141997052","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":"From labs to legislation: The interplay between academic research and political realities","authors":"Saumya Gopalkrishnan Ph.D","doi":"10.1016/j.slasd.2024.100175","DOIUrl":"10.1016/j.slasd.2024.100175","url":null,"abstract":"","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000376/pdfft?md5=74ee6b11020cedb38ee53e6fd094975a&pid=1-s2.0-S2472555224000376-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141908597","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":"Structure-activity relationships of thiadiazole agonists of the human secretin receptor","authors":"Robert Ardecky ,&nbsp;Daniela G. Dengler ,&nbsp;Kaleeckal G. Harikumar ,&nbsp;Mathew M. Abelman ,&nbsp;Jiwen Zou ,&nbsp;Bryan A. Kramer ,&nbsp;Santhi Reddy Ganji ,&nbsp;Steve Olson ,&nbsp;Alina Ly ,&nbsp;Nikhil Puvvula ,&nbsp;Chen-Ting Ma ,&nbsp;Raghuveer Ramachandra ,&nbsp;Eduard A. Sergienko ,&nbsp;Laurence J. Miller","doi":"10.1016/j.slasd.2024.100176","DOIUrl":"10.1016/j.slasd.2024.100176","url":null,"abstract":"<div><p>Agonists of the secretin receptor have potential applications for diseases of the cardiovascular, gastrointestinal, and metabolic systems, yet no clinically-active non-peptidyl agonists of this receptor have yet been developed. In the current work, we have identified a new small molecule lead compound with this pharmacological profile. We have prepared and characterized a systematic structure-activity series around this thiadiazole scaffold to better understand the molecular determinants of its activity. We were able to enhance the <em>in vitro</em> activity and to maintain the specificity of the parent compound. We found the most active candidate to be quite stable in plasma, although it was metabolized by hepatic microsomes. This chemical probe should be useful for <em>in vitro</em> studies and needs to be tested for <em>in vivo</em> pharmacological activity. This could be an important lead toward the development of a first-in-class orally active agonist of the secretin receptor, which could be useful for multiple disease states.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-08-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000388/pdfft?md5=237988fb6dfd70241f35deecdd6ffa90&pid=1-s2.0-S2472555224000388-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141914780","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":"Application of FRET- and BRET-based live-cell biosensors in deorphanization and ligand discovery studies on orphan G protein-coupled receptors","authors":"Joanna J. Sajkowska ,&nbsp;Choi Har Tsang ,&nbsp;Paweł Kozielewicz","doi":"10.1016/j.slasd.2024.100174","DOIUrl":"10.1016/j.slasd.2024.100174","url":null,"abstract":"<div><p>Bioluminescence- and fluorescence-based resonance energy transfer assays have gained considerable attention in pharmacological research as high-throughput scalable tools applicable to drug discovery. To this end, G protein-coupled receptors represent the biggest target class for marketed drugs, and among them, orphan G protein-coupled receptors have the biggest untapped therapeutic potential. In this review, the cases where biophysical methods, BRET and FRET, were employed for deorphanization and ligand discovery studies on orphan G protein-coupled receptors are listed and discussed.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-07-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000364/pdfft?md5=22b61c01ae44de542e774a299be5ac46&pid=1-s2.0-S2472555224000364-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141861891","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 kinetics in drug discovery","authors":"Maria Filipa Pinto ,&nbsp;Julija Sirina ,&nbsp;Nicholas D Holliday ,&nbsp;Claire L McWhirter","doi":"10.1016/j.slasd.2024.100170","DOIUrl":"10.1016/j.slasd.2024.100170","url":null,"abstract":"<div><p>The importance of a drug's kinetic profile and interplay of structure-kinetic activity with PK/PD has long been appreciated in drug discovery. However, technical challenges have often limited detailed kinetic characterization of compounds to the latter stages of projects. This review highlights the advances that have been made in recent years in techniques, instrumentation, and data analysis to increase the throughput of detailed kinetic and mechanistic characterization, enabling its application earlier in the drug discovery process.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000327/pdfft?md5=8c52f41da044edb5c1003b2a3e1004f6&pid=1-s2.0-S2472555224000327-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141536164","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 high-throughput response to the SARS-CoV-2 pandemic","authors":"","doi":"10.1016/j.slasd.2024.100160","DOIUrl":"10.1016/j.slasd.2024.100160","url":null,"abstract":"<div><p>Four years after the beginning of the COVID-19 pandemic, it is important to reflect on the events that have occurred during that time and the knowledge that has been gained. The response to the pandemic was rapid and highly resourced; it was also built upon a foundation of decades of federally funded basic and applied research. Laboratories in government, pharmaceutical, academic, and non-profit institutions all played roles in advancing pre-2020 discoveries to produce clinical treatments. This perspective provides a summary of how the development of high-throughput screening methods in a biosafety level 3 (BSL-3) environment at Southern Research Institute (SR) contributed to pandemic response efforts. The challenges encountered are described, including those of a technical nature as well as those of working under the pressures of an unpredictable virus and pandemic.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000224/pdfft?md5=5e127d48b2c15cd0e588ef0c96314593&pid=1-s2.0-S2472555224000224-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141042759","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 a NanoBRET assay for evaluation of 14-3-3σ molecular glues","authors":"Holly R. Vickery ,&nbsp;Johanna M. Virta ,&nbsp;Markella Konstantinidou,&nbsp;Michelle R. Arkin","doi":"10.1016/j.slasd.2024.100165","DOIUrl":"10.1016/j.slasd.2024.100165","url":null,"abstract":"<div><p>We report the development of a 384-well formatted NanoBRET assay to characterize molecular glues of 14-3-3/client interactions in living cells. The seven isoforms of 14-3-3 are dimeric hub proteins with diverse roles including transcription factor regulation and signal transduction. 14-3-3 interacts with hundreds of client proteins to regulate their function and is therefore an ideal therapeutic target when client selectivity can be achieved. We have developed the NanoBRET system for three 14-3-3σ client proteins CRAF, TAZ, and estrogen receptor α (ERα), which represent three specific binding modes. We have measured stabilization of 14-3-3σ/client complexes by molecular glues with EC₅₀ values between 100 nM and 1 μM in cells, which align with the EC₅₀ values calculated by fluorescence anisotropy in vitro. Developing this NanoBRET system for the hub protein 14-3-3σ allows for a streamlined approach, bypassing multiple optimization steps in the assay development process for other 14-3-3σ clients. The NanoBRET system allows for an assessment of PPI stabilization in a more physiologically relevant, cell-based environment using full-length proteins. The method is applicable to diverse protein-protein interactions (PPIs) and offers a robust platform to explore libraries of compounds for both PPI stabilizers and inhibitors.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000273/pdfft?md5=e9c66b542a3d17a9f5d1ab45060e1740&pid=1-s2.0-S2472555224000273-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141155812","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":"Profiling cells with DELs: Small molecule fingerprinting of cell surfaces","authors":"Jason Deng ,&nbsp;Svetlana Belyanskaya,&nbsp;Ninad Prabhu ,&nbsp;Christopher Arico-Muendel,&nbsp;Hongfeng Deng ,&nbsp;Christopher B. Phelps ,&nbsp;David I. Israel ,&nbsp;Hongfang Yang ,&nbsp;Joseph Boyer ,&nbsp;G. Joseph Franklin ,&nbsp;Jeremy L. Yap ,&nbsp;Kenneth E. Lind ,&nbsp;Ching-Hsuan Tsai ,&nbsp;Christine Donahue ,&nbsp;Jennifer D. Summerfield","doi":"10.1016/j.slasd.2024.100171","DOIUrl":"10.1016/j.slasd.2024.100171","url":null,"abstract":"<div><p>DNA-encoded small molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, it has been used to identify ligands against targets that are soluble or overexpressed on cell surfaces. Here, we report applying cell-based selection methods to profile surfaces of mouse C2C12 myoblasts and myotube cells in an unbiased, target agnostic manner. A panel of on-DNA compounds were identified and confirmed for cell binding selectivity. We optimized the cell selection protocol and employed a novel data analysis method to identify cell selective ligands against a panel of human B and T lymphocytes. We discuss the generality of using this workflow for DNA encoded small molecule library selection and data analysis against different cell types, and the feasibility of applying this method to profile cell surfaces for biomarker and target identification.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":null,"pages":null},"PeriodicalIF":2.7,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000339/pdfft?md5=c0f34f61b83c77e23483474ae6a68680&pid=1-s2.0-S2472555224000339-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141452411","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}