SLAS Discovery最新文献

{"title":"TREM2 hit discovery using temperature-related intensity change (TRIC) technology: A proof-of-concept high-throughput screening approach","authors":"Natalie Fuchs ,&nbsp;Katarzyna Kuncewicz ,&nbsp;Farida El Gaamouch ,&nbsp;Moustafa T. Gabr","doi":"10.1016/j.slasd.2025.100255","DOIUrl":"10.1016/j.slasd.2025.100255","url":null,"abstract":"<div><div>Triggering receptor expressed on myeloid cells 2 (TREM2) is an immunomodulatory receptor implicated in both neurodegenerative diseases and cancer. Depending on the context, TREM2 agonists or inhibitors hold therapeutic potential. To date, the majority of TREM2-targeted strategies have centered on monoclonal antibodies (mAbs), which face limitations such as poor tissue penetration and potential immunogenic side effects. To overcome these challenges and expand the chemical space for TREM2-targeting agents, we developed a high-throughput screening (HTS) platform to identify novel small molecule TREM2 binders. Using temperature-related intensity change (TRIC) technology in a 384-well plate format (NanoTemper Dianthus), we screened two focused compound libraries comprising over 1,200 molecules. From this screen, 18 preliminary hits (1.44 % hit rate) were identified and subsequently validated by dose-response binding studies using microscale thermophoresis (MST), yielding four validated hits (0.32 % hit rate) with binding affinities in the high to medium micromolar range (e.g., <strong>T2337</strong>, <em>K</em>_D = 22.4 µM). The binding of the top hit, <strong>T2337</strong>, was further validated using surface plasmon resonance (SPR). Additionally, we assessed the functional activity of all four validated hits in a cellular assay measuring TREM2-mediated Syk phosphorylation in HEK293 cells co-expressing human TREM2 and its adaptor protein DAP12. These findings establish a robust and scalable platform for the discovery of small molecule TREM2 modulators and serve as a proof-of-concept for broader HTS campaigns targeting TREM2.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100255"},"PeriodicalIF":2.7,"publicationDate":"2025-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144763963","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":"Off-target effects of oligonucleotides and approaches of preclinical assessments","authors":"Haiwen Ruan ,&nbsp;Dehu Dou ,&nbsp;Jing Lu ,&nbsp;Xia Xiao ,&nbsp;Xinjiang Gong ,&nbsp;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":"2025-07-27","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}

{"title":"Bonferroni’s method, not Tukey’s, should be used to control the total number of false positives when making multiple pairwise comparisons in experiments with few replicates","authors":"Adam Zweifach","doi":"10.1016/j.slasd.2025.100253","DOIUrl":"10.1016/j.slasd.2025.100253","url":null,"abstract":"<div><div>Statistical tests can be used to help determine whether experimental manipulations produce effects. In tests of means, when more than two groups are compared the total number of Type 1 errors (false positive results) increases unless a correction is used. Tukey’s method is thought to offer good control of the number of false positives and high statistical power when all pairwise comparisons are made. However, the number of replicates in laboratory experiments is often quite low, and small sample sizes can undermine assumptions underlying statistical methods. I used simulations to investigate how well ANOVA followed by different post-hoc tests controls the total number of false positives when there are 3- 6 experimental groups and 2- 6 experimental replicates, conditions that span the range of typical values. Tukey’s method, one of the most common, allows too many. I investigated 11 other approaches to controlling false positives and found that none is as effective as the simple Bonferroni correction or offers much more power. I conclude that researchers should not make all pairwise comparisons using ANOVA followed by Tukey’s method but instead use Bonferroni’s method on a limited number of pre-selected comparisons.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100253"},"PeriodicalIF":2.7,"publicationDate":"2025-07-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144700594","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":"Overcoming a false-positive mechanism in RapidFire MRM-based high throughput screening","authors":"De Lin ,&nbsp;Lesley-Anne Pearson ,&nbsp;Shamshad Ahmad,&nbsp;Sandra O’Neill,&nbsp;John Post,&nbsp;Colin Robinson,&nbsp;Duncan E. Scott,&nbsp;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":"2025-07-18","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}

{"title":"An enzyme activity-based workflow for the identification and characterization of covalent inhibitors","authors":"Buyun Tang,&nbsp;Tatiana Gladysheva,&nbsp;Paul Lang","doi":"10.1016/j.slasd.2025.100250","DOIUrl":"10.1016/j.slasd.2025.100250","url":null,"abstract":"<div><div>The field of covalent drug development has advanced rapidly, offering promising therapeutic potential due to the ability of these drugs to form slowly reversible or irreversible bonds with target proteins, resulting in prolonged pharmacodynamic effects. This distinctive mechanism of action has sparked resurging interest in covalent inhibitors across various disease areas, including oncology, neurological disorders, and infectious diseases. However, characterization of covalent inhibitors poses unique challenges, highlighting the need for simplified and robust assay methods. This protocol describes an enzyme activity-based workflow designed to identify and characterize covalent inhibitors efficiently. By streamlining the evaluation process, this approach enhances the reliability and reproducibility of covalent inhibitor assessment, ultimately accelerating the discovery and optimization of novel covalent therapeutics.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100250"},"PeriodicalIF":2.7,"publicationDate":"2025-07-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144676733","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":"Nanobodies: A new frontier in antiviral therapies","authors":"Ahmed Mohammed ,&nbsp;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":"2025-07-17","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}

{"title":"Time resolved cell painting enables rapid assessment of cell phenotypes","authors":"Franziska A. Hecker ,&nbsp;Bruno Leggio ,&nbsp;Tim Koenig ,&nbsp;Karsten Niehaus ,&nbsp;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":"2025-07-09","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}

{"title":"A scalable human gut-immune co-culture model for evaluating inflammatory bowel disease anti-inflammatory therapies","authors":"Swetha Peddibhotla ,&nbsp;Lauren A. Boone ,&nbsp;Earnest L. Taylor,&nbsp;Bryan E. McQueen,&nbsp;Elizabeth M. Boazak","doi":"10.1016/j.slasd.2025.100248","DOIUrl":"10.1016/j.slasd.2025.100248","url":null,"abstract":"<div><div>Current treatments for inflammatory bowel disease (IBD) are often ineffective long-term, as many patients ultimately become unresponsive to anti-inflammatory drugs. The need for improved therapeutics is urgent. Animal models utilized for drug development are limited by interspecies variability and poor translatability. However, most in vitro models lack the sophistication to model the key interplay of the immune system with the intestinal epithelium in line with the known role of the immune system in the etiology of the disease.</div><div>To address this gap, we developed a primary intestinal epithelial cell co-culture system to incorporate elements of innate immune signaling. This system models immune-epithelial interactions using RepliGut^Ⓡ - Planar Transverse Colon cultured on a Transwell™ system with THP-1 derived macrophages in a receiver compartment of a 96-well plate format, compatible with high-throughput screening (HTS) workflows. Epithelial barrier integrity and cell viability were maintained in co-culture with unstimulated macrophages. However, similar to the pathology associated with IBD, epithelial integrity was compromised in co-culture with LPS + IFN-γ pre-stimulated macrophages as evidenced by declining TEER and cell viability and increased inflammatory cytokine release. Cotreatment with anti-inflammatory IBD therapeutics adalimumab or tofacitinib mitigated these effects, demonstrating the model’s ability to replicate key inflammatory responses and prevention.</div><div>Reproducibility and scalability of the model system further position the model for screening and/or mechanistic interrogation of anti-inflammatory drugs, improving drug discovery, and accelerating the translation of new IBD therapies into clinical practice.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100248"},"PeriodicalIF":2.7,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144621451","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 the identification of dual inhibitors of BRD4 and RIPK3 toward the development of small-molecule medical countermeasure agents against arsenicals","authors":"Marina Fosso Yatchang ,&nbsp;Ling Zhai ,&nbsp;Omar Moukha-Chafiq ,&nbsp;Bini Mathew ,&nbsp;Fuad Al Abir ,&nbsp;Sixue Zhang ,&nbsp;Pedro Ruiz ,&nbsp;Sara McKellip ,&nbsp;Miranda Nebane ,&nbsp;Jake Y. Chen ,&nbsp;Anupam Agarwal ,&nbsp;James R. Bostwick ,&nbsp;Mark J. Suto ,&nbsp;Mohammad Athar ,&nbsp;Corinne E. Augelli-Szafran","doi":"10.1016/j.slasd.2025.100247","DOIUrl":"10.1016/j.slasd.2025.100247","url":null,"abstract":"<div><div>Warfare arsenicals are potent blistering agents and cause severe inflammation following their skin exposure. Data from our group (unpublished) show that these chemicals act by activating bromodomain-4 and RIPK signaling. To develop a dual inhibitor of the bromodomain-containing protein 4 (BRD4) and the receptor-interacting serine/threonine-protein kinase 3 (RIPK3), we conducted a high-throughput screening (HTS) campaign for inhibitors of BRD4 and RIPK3 activity to identify anti-inflammatory agent candidates that could alleviate arsenicals-induced injury. Our primary assays were adapted to 384-well microplates and used to screen a collection of 4074 compounds consisting of FDA-approved drugs and other bioactive compounds. The BRD4 primary screen had an average Z' value of 0.93 and a signal-to-background (S/B) ratio of 3018, while the RIPK3 primary screen had an average Z' value of 0.86 and S/<em>B</em> = 12.6. A counter screen assay was used to ensure activity was due to target engagement and not assay interference. Hits that inhibited BRD4 binding by &gt; 54.6 % and kinase activity by &gt; 22.4 % in the primary screen and were not statistical outliers in the counter screen assays, were confirmed in concentration-response format. Hits were also tested in a cell-based IL-6 assay to determine corresponding inflammatory inhibitory activity. Eighteen compounds were active in both BRD4 and RIPK3 assays, of which three displayed IC₅₀ values &lt; 10 μM with promising IL-6 inhibition. These compounds could serve as good candidates for further chemical optimization for the development of small-molecule medical counter measure agents against arsenicals.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"35 ","pages":"Article 100247"},"PeriodicalIF":2.7,"publicationDate":"2025-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144565499","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 human tissue model for respiratory viruses: Automating the use of human airway epithelial tissues for faster drug discovery","authors":"N. Miranda Nebane,&nbsp;Andrew Reece,&nbsp;Lynn Rasmussen,&nbsp;Melinda Sosa,&nbsp;Pedro Ruiz,&nbsp;Sara M. Cabrera,&nbsp;Yohanka Martinez-Gzegozewska,&nbsp;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 (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 &gt; 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":"2025-06-26","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}