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.
{"title":"The role of m6A in lipid metabolism-related diseases","authors":"Qianhui Zeng , Yuxuan Yang , Yinquan Zhang , Siwen Jiang","doi":"10.1016/j.slasd.2025.100257","DOIUrl":"10.1016/j.slasd.2025.100257","url":null,"abstract":"<div><div>N6-methyladenosine (m<sup>6</sup>A) 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 m<sup>6</sup>A methyltransferases, demethylases, and recognition proteins, thereby influencing a wide array of biological processes. Notably, the significance of m<sup>6</sup>A 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 m<sup>6</sup>A 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 m<sup>6</sup>A regulatory factors. This work provides theoretical references for the development of future therapeutic strategies targeting m<sup>6</sup>A 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}
Pub Date : 2025-09-01Epub Date: 2025-07-17DOI: 10.1016/j.slasd.2025.100251
Ahmed Mohammed , Mujahed I. Muustafa
Nanobodies, derived from the immune systems of camelids such as alpacas and llamas, represent a novel class of therapeutics with significant potential in fighting respiratory viral infections, such as SARS-CoV-2 and influenza. Nanobodies are small biomolecules that are highly stable and have unique binding features that allow for the effective neutralization of viral particles and inhibition of viral replication. This review highlights the advantages of nanobodies over traditional antibodies, including cost-effective production and enhanced specificity for target antigens. We discuss the mechanisms through which nanobodies block viral entry, their applications in diagnostics, and the methodologies for their development, such as phage display technology. Furthermore, we explore the efficacy of nanobodies in preclinical studies and their potential in clinical settings. As research progresses, structural optimization and the exploration of combination therapies may enhance their therapeutic efficacy, providing a promising approach for addressing global health challenges caused by emerging viral pathogens.
{"title":"Nanobodies: A new frontier in antiviral therapies","authors":"Ahmed Mohammed , Mujahed I. Muustafa","doi":"10.1016/j.slasd.2025.100251","DOIUrl":"10.1016/j.slasd.2025.100251","url":null,"abstract":"<div><div>Nanobodies, derived from the immune systems of camelids such as alpacas and llamas, represent a novel class of therapeutics with significant potential in fighting respiratory viral infections, such as SARS-CoV-2 and influenza. Nanobodies are small biomolecules that are highly stable and have unique binding features that allow for the effective neutralization of viral particles and inhibition of viral replication. This review highlights the advantages of nanobodies over traditional antibodies, including cost-effective production and enhanced specificity for target antigens. We discuss the mechanisms through which nanobodies block viral entry, their applications in diagnostics, and the methodologies for their development, such as phage display technology. Furthermore, we explore the efficacy of nanobodies in preclinical studies and their potential in clinical settings. As research progresses, structural optimization and the exploration of combination therapies may enhance their therapeutic efficacy, providing a promising approach for addressing global health challenges caused by emerging viral pathogens.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100251"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144669122","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-08-09DOI: 10.1016/j.slasd.2025.100258
Francisco Castillo , Thomas A. Mackenzie , Elisabeth Domingo , Inmaculada Iañez , Matthew B. Robers , Jennifer Wilkinson , Erika Kay-Tsumagari , Martha O’Brien , Olga Genilloud , Rosario Fernandez-Godino , Maria C. Ramos
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.
{"title":"Identification of novel inflammasome inhibitors via cellular NLRP3 target engagement assays","authors":"Francisco Castillo , Thomas A. Mackenzie , Elisabeth Domingo , Inmaculada Iañez , Matthew B. Robers , Jennifer Wilkinson , Erika Kay-Tsumagari , Martha O’Brien , Olga Genilloud , Rosario Fernandez-Godino , 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":"2025-09-01","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}
Pub Date : 2025-09-01Epub Date: 2025-08-12DOI: 10.1016/j.slasd.2025.100262
Emery Smith , Qibin Geng , Justin Shumate , Yuka Otsuka , Louis Scampavia , Thomas D. Bannister , Timothy P. Spicer
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.
{"title":"High throughput screening for SARS-CoV-2 inhibitors targeting 5 helix bundle","authors":"Emery Smith , Qibin Geng , Justin Shumate , Yuka Otsuka , Louis Scampavia , Thomas D. Bannister , 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":"2025-09-01","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}
Drug discovery and development have been a significant focus of medicinal and pharmaceutical research, continually striving to meet the growing challenges posed by complex diseases and medical conditions. In drug development, quantum dots (QDs) can be utilized in fluorescent assays for drug discovery and as fluorescent labels in drug delivery systems to monitor the metabolism of drugs in the body. As efforts to unravel the mysteries of human health and design innovative therapeutic solutions increase, the roles of model organisms in advancing understanding and accelerating discovery and development are also expanding. Zebrafish (Danio rerio) have emerged as a prominent model organism in the field of drug screening and development due to their unique biological attributes and experimental advantages. Many pharmaceutical products and drugs developed in the pharmaceutical industry fail in clinical trials due to unanticipated toxic side effects. Similarly, despite the interesting characteristics and versatile applications of QDs in drug development, there are a limited number of clinical trials involving QDs, hindered by complex pharmaceutical, industrial, technical, and biological challenges such as toxicity. Therefore, this article aims to highlight the importance of using zebrafish embryos and eleutheroembryos models for the toxicological assessment of pharmaceutical drugs and QDs in drug delivery and development. This review summarizes the developments available in the literature regarding the evaluation of the toxicity of QDs and drugs using zebrafish assays. The use of zebrafish models for safety profiling and pharmacological preclinical screening of pharmaceutical drugs and QDs will provide more insights than cellular assays and offer valuable information for mammalian experiments.
{"title":"Toxicity evaluation of pharmaceutical drugs and quantum dots (QDs) using zebrafish embryos – A comprehensive review","authors":"Motunrayo Faderera Adegoke , Olamide Abiodun Daramola , Kayode Omotayo Adeniyi , Madan Poka , Patrick Hulisani Demana , Xavier Siwe Noundou","doi":"10.1016/j.slasd.2025.100241","DOIUrl":"10.1016/j.slasd.2025.100241","url":null,"abstract":"<div><div>Drug discovery and development have been a significant focus of medicinal and pharmaceutical research, continually striving to meet the growing challenges posed by complex diseases and medical conditions. In drug development, quantum dots (QDs) can be utilized in fluorescent assays for drug discovery and as fluorescent labels in drug delivery systems to monitor the metabolism of drugs in the body. As efforts to unravel the mysteries of human health and design innovative therapeutic solutions increase, the roles of model organisms in advancing understanding and accelerating discovery and development are also expanding. Zebrafish (Danio rerio) have emerged as a prominent model organism in the field of drug screening and development due to their unique biological attributes and experimental advantages. Many pharmaceutical products and drugs developed in the pharmaceutical industry fail in clinical trials due to unanticipated toxic side effects. Similarly, despite the interesting characteristics and versatile applications of QDs in drug development, there are a limited number of clinical trials involving QDs, hindered by complex pharmaceutical, industrial, technical, and biological challenges such as toxicity. Therefore, this article aims to highlight the importance of using zebrafish embryos and eleutheroembryos models for the toxicological assessment of pharmaceutical drugs and QDs in drug delivery and development. This review summarizes the developments available in the literature regarding the evaluation of the toxicity of QDs and drugs using zebrafish assays. The use of zebrafish models for safety profiling and pharmacological preclinical screening of pharmaceutical drugs and QDs will provide more insights than cellular assays and offer valuable information for mammalian experiments.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100241"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144230301","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-07-18DOI: 10.1016/j.slasd.2025.100252
De Lin , Lesley-Anne Pearson , Shamshad Ahmad, Sandra O’Neill, John Post, Colin Robinson, Duncan E. Scott, Ian H. Gilbert
False-positives plague high-throughput screening in general and are costly as they consume resource and time to resolve. Methods that can rapidly identify such compounds at the initial screen are therefore of great value. Advances in mass spectrometry have led to the ability to screen inhibitors in drug discovery applications by direct detection of an enzyme reaction product. The technique is free from some of the artefacts that trouble classical assays such as fluorescence interference. Its direct nature negates the need for coupling enzymes and hence is simpler with fewer opportunities for artefacts. Despite its myriad advantages, we report here a mechanism for false-positive hits which has not been reported in the literature. Further we have developed a pipeline for detecting these false-positive hits and suggest a method to mitigate against them.
{"title":"Overcoming a false-positive mechanism in RapidFire MRM-based high throughput screening","authors":"De Lin , Lesley-Anne Pearson , Shamshad Ahmad, Sandra O’Neill, John Post, Colin Robinson, Duncan E. Scott, Ian H. Gilbert","doi":"10.1016/j.slasd.2025.100252","DOIUrl":"10.1016/j.slasd.2025.100252","url":null,"abstract":"<div><div>False-positives plague high-throughput screening in general and are costly as they consume resource and time to resolve. Methods that can rapidly identify such compounds at the initial screen are therefore of great value. Advances in mass spectrometry have led to the ability to screen inhibitors in drug discovery applications by direct detection of an enzyme reaction product. The technique is free from some of the artefacts that trouble classical assays such as fluorescence interference. Its direct nature negates the need for coupling enzymes and hence is simpler with fewer opportunities for artefacts. Despite its myriad advantages, we report here a mechanism for false-positive hits which has not been reported in the literature. Further we have developed a pipeline for detecting these false-positive hits and suggest a method to mitigate against them.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100252"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144676734","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-06-16DOI: 10.1016/j.slasd.2025.100243
Lan Mi , Mingxu You
Fluorescence- and bioluminescence-based probes are valuable tools for understanding cell functions in health and disease. Bioluminescence offers an ideal complementary readout to fluorescence due to its minimal background interference and self-illuminating nature. We previously introduced the first type of genetically encodable RNA-based bioluminescence resonance energy transfer (BRET) sensors. These RNA-based probes are highly programmable and can be modularly engineered to detect various cellular targets. While this system was successfully validated in vitro and from the entire cell population within a microplate, the BRET signals were quite dim and difficult to visualize at the single-cell level under a microscope. The ability of single-cell bioluminescence imaging is critical for studying cell-to-cell variations and spatiotemporal changes of cellular targets in different signaling pathways or upon drug treatment. In this study, we will introduce strategies that can enhance the functionality and capability of RNA-based BRET sensors for real-time cellular imaging and sensing. Using commonly used widefield microscopes, single-cell bioluminescent detection of various metabolites and other small molecules can be achieved in both bacterial and mammalian cells. This advancement represents a significant step toward the future development of genetically encoded RNA-based bioluminescent tools for studying disease mechanisms, high-throughput drug screening, and in vivo imaging.
{"title":"Genetically encoded fluorogenic RNA-based bioluminescence resonance energy transfer (BRET) sensors for cellular imaging and target detection","authors":"Lan Mi , Mingxu You","doi":"10.1016/j.slasd.2025.100243","DOIUrl":"10.1016/j.slasd.2025.100243","url":null,"abstract":"<div><div>Fluorescence- and bioluminescence-based probes are valuable tools for understanding cell functions in health and disease. Bioluminescence offers an ideal complementary readout to fluorescence due to its minimal background interference and self-illuminating nature. We previously introduced the first type of genetically encodable RNA-based bioluminescence resonance energy transfer (BRET) sensors. These RNA-based probes are highly programmable and can be modularly engineered to detect various cellular targets. While this system was successfully validated <em>in vitro</em> and from the entire cell population within a microplate, the BRET signals were quite dim and difficult to visualize at the single-cell level under a microscope. The ability of single-cell bioluminescence imaging is critical for studying cell-to-cell variations and spatiotemporal changes of cellular targets in different signaling pathways or upon drug treatment. In this study, we will introduce strategies that can enhance the functionality and capability of RNA-based BRET sensors for real-time cellular imaging and sensing. Using commonly used widefield microscopes, single-cell bioluminescent detection of various metabolites and other small molecules can be achieved in both bacterial and mammalian cells. This advancement represents a significant step toward the future development of genetically encoded RNA-based bioluminescent tools for studying disease mechanisms, high-throughput drug screening, and <em>in vivo</em> imaging.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100243"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144328043","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-06-26DOI: 10.1016/j.slasd.2025.100246
N. Miranda Nebane, Andrew Reece, Lynn Rasmussen, Melinda Sosa, Pedro Ruiz, Sara M. Cabrera, Yohanka Martinez-Gzegozewska, Paige Vinson
Animal testing for drug discovery is expensive and the decision to test a compound in an in vivo model should be carefully considered. In addition, the FDA Modernization Act has resulted in the allowance of alternatives to animal models for testing the safety and efficacy of drug candidates. Among these alternatives are human tissue models that provide a human-relevant context. Specialized cell types can be produced from primary human cells and used for basic research and drug discovery purposes. One of these is a 3D model for respiratory disease research, consisting of human-derived tracheal/bronchial epithelial cells. Though this translational Human Airway Epithelial (HAE) model is currently being employed by many researchers, this work is primarily done using individual tissue inserts placed in 6-well plates. This low-throughput approach is labor-intensive, time-consuming and expensive (high cost per each compound screened). We have established a high-throughput HAE assay that can be used for compound screening to advance respiratory virus programs. This is significant as it allows a higher number of compounds to be evaluated before being tested in vivo, allowing a more comprehensive comparison of candidates at this later discovery stage. We developed 96-well assays to evaluate compounds for Influenza, Respiratory Syncytial Virus (RSV) and Coronaviruses (including SARS-CoV-2), and these assays are easily adaptable to other respiratory viruses like Human metapneumovirus (HMPV). The development process involved performing a titration of each virus for 50 % tissue culture infectious dose (TCID50) calculations and determining the optimal HAE infection time in a time course experiment, with every step of the process automated to increase speed and precision. Following infection of the HAE tissues, the amount of infectious virus in apically collected sample was assessed in a Cytopathic Effect (CPE) or Virus Titer Reduction (VTR) assay in an appropriate cell line for that particular virus. The optimized assays consistently showed Z’ values > 0.75 and were used to test reference compounds relevant to each antiviral assay. Potency values for oseltamivir and ribavirin against H3N2 A/Udorn/72 were 100 nM and 5.3 µM, respectively. The streamlined assay development process highlights the benefits of miniaturizing HAE assays from individual tissue inserts placed inside a 6-well plate to a 96-well format, providing a high-throughput solution for human 3D in vitro respiratory tissue models.
{"title":"A high-throughput human tissue model for respiratory viruses: Automating the use of human airway epithelial tissues for faster drug discovery","authors":"N. Miranda Nebane, Andrew Reece, Lynn Rasmussen, Melinda Sosa, Pedro Ruiz, Sara M. Cabrera, Yohanka Martinez-Gzegozewska, Paige Vinson","doi":"10.1016/j.slasd.2025.100246","DOIUrl":"10.1016/j.slasd.2025.100246","url":null,"abstract":"<div><div>Animal testing for drug discovery is expensive and the decision to test a compound in an in vivo model should be carefully considered. In addition, the FDA Modernization Act has resulted in the allowance of alternatives to animal models for testing the safety and efficacy of drug candidates. Among these alternatives are human tissue models that provide a human-relevant context. Specialized cell types can be produced from primary human cells and used for basic research and drug discovery purposes. One of these is a 3D model for respiratory disease research, consisting of human-derived tracheal/bronchial epithelial cells. Though this translational Human Airway Epithelial (HAE) model is currently being employed by many researchers, this work is primarily done using individual tissue inserts placed in 6-well plates. This low-throughput approach is labor-intensive, time-consuming and expensive (high cost per each compound screened). We have established a high-throughput HAE assay that can be used for compound screening to advance respiratory virus programs. This is significant as it allows a higher number of compounds to be evaluated before being tested in vivo, allowing a more comprehensive comparison of candidates at this later discovery stage. We developed 96-well assays to evaluate compounds for Influenza, Respiratory Syncytial Virus (RSV) and Coronaviruses (including SARS-CoV-2), and these assays are easily adaptable to other respiratory viruses like Human metapneumovirus (HMPV). The development process involved performing a titration of each virus for 50 % tissue culture infectious dose (TCID<sub>50</sub>) calculations and determining the optimal HAE infection time in a time course experiment, with every step of the process automated to increase speed and precision. Following infection of the HAE tissues, the amount of infectious virus in apically collected sample was assessed in a Cytopathic Effect (CPE) or Virus Titer Reduction (VTR) assay in an appropriate cell line for that particular virus. The optimized assays consistently showed Z’ values > 0.75 and were used to test reference compounds relevant to each antiviral assay. Potency values for oseltamivir and ribavirin against H3N2 A/Udorn/72 were 100 nM and 5.3 µM, respectively. The streamlined assay development process highlights the benefits of miniaturizing HAE assays from individual tissue inserts placed inside a 6-well plate to a 96-well format, providing a high-throughput solution for human 3D in vitro respiratory tissue models.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100246"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144517622","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-07-27DOI: 10.1016/j.slasd.2025.100254
Haiwen Ruan , Dehu Dou , Jing Lu , Xia Xiao , Xinjiang Gong , Xuefeng Zhang
Oligonucleotide-based therapies, such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), represent a class of therapeutic agents that specifically target gene transcription or translation mechanisms through sequence specificity. These pharmaceuticals exhibit significant promise in the treatment of genetic disorders, including spinal muscular atrophy, as well as malignancies, viral infections, and metabolic diseases. Nonetheless, unintended toxicity continues to pose a considerable challenge and remain a critical safety concern in the development of oligonucleotide therapeutics (ONTs). Off-target toxicity may be caused by hybridization to sequences that are similar but not identical to the target, hybridization-independent sequence related, or sequence- and hybridization-independent effects. The effects may result in diminished transcript levels, decreased translation rates, or anomalous splicing, employing same molecular pathways and protein machinery as the desired on-target effects. Currently, there exists no established methodology for the systematic identification and evaluation of off-target toxicity, which may hinder the optimization of safety approaches. This review delineates significant nonclinical toxicities and clinical adverse effects by summarizing and analyzing approved oligonucleotides with their off-target assays, encompassing the limitations of nonclinical off-target effects and the potential off-target mechanisms. Plus, it discusses and emphasizes the factors that lead to the off target of ONTs, systematically offers approaches and workflows of preclinical assessments to enhance the transfer value of oligonucleotide therapies from nonclinical to clinical trials by managing unavoidable off-target effects.
{"title":"Off-target effects of oligonucleotides and approaches of preclinical assessments","authors":"Haiwen Ruan , Dehu Dou , Jing Lu , Xia Xiao , Xinjiang Gong , Xuefeng Zhang","doi":"10.1016/j.slasd.2025.100254","DOIUrl":"10.1016/j.slasd.2025.100254","url":null,"abstract":"<div><div>Oligonucleotide-based therapies, such as antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), represent a class of therapeutic agents that specifically target gene transcription or translation mechanisms through sequence specificity. These pharmaceuticals exhibit significant promise in the treatment of genetic disorders, including spinal muscular atrophy, as well as malignancies, viral infections, and metabolic diseases. Nonetheless, unintended toxicity continues to pose a considerable challenge and remain a critical safety concern in the development of oligonucleotide therapeutics (ONTs). Off-target toxicity may be caused by hybridization to sequences that are similar but not identical to the target, hybridization-independent sequence related, or sequence- and hybridization-independent effects. The effects may result in diminished transcript levels, decreased translation rates, or anomalous splicing, employing same molecular pathways and protein machinery as the desired on-target effects. Currently, there exists no established methodology for the systematic identification and evaluation of off-target toxicity, which may hinder the optimization of safety approaches. This review delineates significant nonclinical toxicities and clinical adverse effects by summarizing and analyzing approved oligonucleotides with their off-target assays, encompassing the limitations of nonclinical off-target effects and the potential off-target mechanisms. Plus, it discusses and emphasizes the factors that lead to the off target of ONTs, systematically offers approaches and workflows of preclinical assessments to enhance the transfer value of oligonucleotide therapies from nonclinical to clinical trials by managing unavoidable off-target effects.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100254"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144721301","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-09-01Epub Date: 2025-07-09DOI: 10.1016/j.slasd.2025.100249
Franziska A. Hecker , Bruno Leggio , Tim Koenig , Karsten Niehaus , Sven Geibel
Cell Painting is an advanced imaging technique for drug discovery used to study cellular phenotypes by simultaneously labeling various organelles/structures and analyzing the resulting multidimensional phenotypic features through a sophisticated data analysis pipeline. Based on established phenotyping methodologies, this method has relied on incubation times of typically around 48 h for the assessment of phenotypic fingerprints. Here we provide evidence that earlier assessments show more robust results with increased significance of phenotypic fingerprints that better reflect primary physiological effects.
Our study included compounds that range from representatives with modes of action that result in immediate phenotypic changes, such as energy metabolism inhibitors, to representatives that typically show pronounced phenotypes after several days, such as developmental inhibitors. Remarkably, we observed that for all compounds, primary cellular alterations were best detected at early timepoints after treatment, specifically at 6 h for Sf9 insect cells and shortly later timepoints for mammalian U2OS cells. Brief incubation periods enable the capture of primary effects of treatments while minimizing the influence of secondary changes as well as downstream phenotypic alterations like, for example, cell death. This enhances the specificity and accuracy of Cell Painting and consequently provides a more immediate depiction of primary actions from compounds. Notably, it also improves the efficiency of experimental workflows.
In conclusion, we propose a more rapid assessment of cell phenotypes and morphology in the Cell Painting assay to enable a higher throughput in drug discovery screenings.
{"title":"Time resolved cell painting enables rapid assessment of cell phenotypes","authors":"Franziska A. Hecker , Bruno Leggio , Tim Koenig , Karsten Niehaus , Sven Geibel","doi":"10.1016/j.slasd.2025.100249","DOIUrl":"10.1016/j.slasd.2025.100249","url":null,"abstract":"<div><div>Cell Painting is an advanced imaging technique for drug discovery used to study cellular phenotypes by simultaneously labeling various organelles/structures and analyzing the resulting multidimensional phenotypic features through a sophisticated data analysis pipeline. Based on established phenotyping methodologies, this method has relied on incubation times of typically around 48 h for the assessment of phenotypic fingerprints. Here we provide evidence that earlier assessments show more robust results with increased significance of phenotypic fingerprints that better reflect primary physiological effects.</div><div>Our study included compounds that range from representatives with modes of action that result in immediate phenotypic changes, such as energy metabolism inhibitors, to representatives that typically show pronounced phenotypes after several days, such as developmental inhibitors. Remarkably, we observed that for all compounds, primary cellular alterations were best detected at early timepoints after treatment, specifically at 6 h for Sf9 insect cells and shortly later timepoints for mammalian U2OS cells. Brief incubation periods enable the capture of primary effects of treatments while minimizing the influence of secondary changes as well as downstream phenotypic alterations like, for example, cell death. This enhances the specificity and accuracy of Cell Painting and consequently provides a more immediate depiction of primary actions from compounds. Notably, it also improves the efficiency of experimental workflows.</div><div>In conclusion, we propose a more rapid assessment of cell phenotypes and morphology in the Cell Painting assay to enable a higher throughput in drug discovery screenings.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100249"},"PeriodicalIF":2.7,"publicationDate":"2025-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144621452","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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