Pub Date : 2025-01-16DOI: 10.1016/j.slasd.2025.100214
Martin P. Schwalm , Stefan Knapp
Cancer research, cancer treatment, and the field of chemical biology are examples which heavily rely on the discovery of selective kinase inhibitors. While determining on-target potency is often feasible for most laboratories, the equally critical but frequently neglected selectivity screening remains less accessible to the broader scientific community. This limitation can stem from various factors, such as the unavailability of a large number of purified kinases or the costs associated with commercial screening systems. To address these challenges and enable a wider range of scientists, this protocol leverages a commercial kinome selectivity screen to facilitate a low-cost, two-day, single-plate selectivity screen against 192 kinases.
{"title":"Single-plate kinome screening in live-cells to enable highly cost-efficient kinase inhibitor profiling","authors":"Martin P. Schwalm , Stefan Knapp","doi":"10.1016/j.slasd.2025.100214","DOIUrl":"10.1016/j.slasd.2025.100214","url":null,"abstract":"<div><div>Cancer research, cancer treatment, and the field of chemical biology are examples which heavily rely on the discovery of selective kinase inhibitors. While determining on-target potency is often feasible for most laboratories, the equally critical but frequently neglected selectivity screening remains less accessible to the broader scientific community. This limitation can stem from various factors, such as the unavailability of a large number of purified kinases or the costs associated with commercial screening systems. To address these challenges and enable a wider range of scientists, this protocol leverages a commercial kinome selectivity screen to facilitate a low-cost, two-day, single-plate selectivity screen against 192 kinases.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100214"},"PeriodicalIF":2.7,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143017975","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}
Pub Date : 2025-01-16DOI: 10.1016/j.slasd.2025.100213
Doaa S․R․ Khafaga , Ghazala Muteeb , Darin․W․ Aswa , Mohammad Aatif , Mohd Farhan , Salma Allam
Brain metastasis (BM) from colon cancer is associated with a poor prognosis and restricted treatment alternatives, largely due to issues related to blood-brain barrier (BBB) permeability and the negative effects of standard chemotherapy. Nanotechnology improves treatment efficacy by enabling targeted and controlled drug delivery. This review article evaluates the potential of nanotechnology-based therapies for treating colon cancer BM, emphasizing their capacity to cross the BBB, diminish metastatic growth, and enhance overall survival rates. A review of multiple studies evaluated nanoparticles (NPs) as carriers for chemotherapy, focusing on parameters including particle size, surface charge, and drug-loading capacity. The study also reviewed studies that examined BBB penetration, in vitro tumor accumulation, and in vivo tumor growth inhibition. In vitro findings indicated that NPs accumulate more efficiently in BM tissue than in healthy brain tissue and show significant BBB penetration. In vivo, nanotherapy markedly inhibited tumor growth and prolonged survival relative to conventional chemotherapy or control treatments while also exhibiting reduced side effects. Recent studies demonstrated that plant extracts can effectively and safely synthesize nanomaterials, positioning them as a viable and environmentally friendly precursor for nanomaterial production. Nanotechnology-based therapies demonstrate significant potential in the treatment of colon cancer BM by minimizing systemic toxicity, enhancing therapeutic efficacy, and facilitating more targeted drug delivery. Further research is required to confirm these findings and implement them in clinical practice.
{"title":"Green chemistry: Modern therapies using nanocarriers for treating rare brain cancer metastasis from colon cancer","authors":"Doaa S․R․ Khafaga , Ghazala Muteeb , Darin․W․ Aswa , Mohammad Aatif , Mohd Farhan , Salma Allam","doi":"10.1016/j.slasd.2025.100213","DOIUrl":"10.1016/j.slasd.2025.100213","url":null,"abstract":"<div><div>Brain metastasis (BM) from colon cancer is associated with a poor prognosis and restricted treatment alternatives, largely due to issues related to blood-brain barrier (BBB) permeability and the negative effects of standard chemotherapy. Nanotechnology improves treatment efficacy by enabling targeted and controlled drug delivery. This review article evaluates the potential of nanotechnology-based therapies for treating colon cancer BM, emphasizing their capacity to cross the BBB, diminish metastatic growth, and enhance overall survival rates. A review of multiple studies evaluated nanoparticles (NPs) as carriers for chemotherapy, focusing on parameters including particle size, surface charge, and drug-loading capacity. The study also reviewed studies that examined BBB penetration, <em>in vitro</em> tumor accumulation, and <em>in vivo</em> tumor growth inhibition. <em>In vitro</em> findings indicated that NPs accumulate more efficiently in BM tissue than in healthy brain tissue and show significant BBB penetration. <em>In vivo</em>, nanotherapy markedly inhibited tumor growth and prolonged survival relative to conventional chemotherapy or control treatments while also exhibiting reduced side effects. Recent studies demonstrated that plant extracts can effectively and safely synthesize nanomaterials, positioning them as a viable and environmentally friendly precursor for nanomaterial production. Nanotechnology-based therapies demonstrate significant potential in the treatment of colon cancer BM by minimizing systemic toxicity, enhancing therapeutic efficacy, and facilitating more targeted drug delivery. Further research is required to confirm these findings and implement them in clinical practice.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100213"},"PeriodicalIF":2.7,"publicationDate":"2025-01-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143017967","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}
Pub Date : 2025-01-15DOI: 10.1016/j.slasd.2025.100209
Lauren Toms, Lorna FitzPatrick, Philip Auckland
At the turn of the century a fundamental resolution barrier in fluorescence microscopy known as the diffraction limit was broken, giving rise to the field of super-resolution microscopy. Subsequent nanoscopic investigation with visible light revolutionised our understanding of how previously unknown molecular features give rise to the emergent behaviour of cells. It transpires that the devil is in these fine molecular details, and essential nanoscale processes were found everywhere researchers chose to look. Now, after nearly two decades, super-resolution microscopy has begun to address previously unmet challenges in the study of human disease and is poised to become a pivotal tool in drug discovery.
{"title":"Super-resolution microscopy as a drug discovery tool","authors":"Lauren Toms, Lorna FitzPatrick, Philip Auckland","doi":"10.1016/j.slasd.2025.100209","DOIUrl":"10.1016/j.slasd.2025.100209","url":null,"abstract":"<div><div>At the turn of the century a fundamental resolution barrier in fluorescence microscopy known as the diffraction limit was broken, giving rise to the field of super-resolution microscopy. Subsequent nanoscopic investigation with visible light revolutionised our understanding of how previously unknown molecular features give rise to the emergent behaviour of cells. It transpires that the devil is in these fine molecular details, and essential nanoscale processes were found everywhere researchers chose to look. Now, after nearly two decades, super-resolution microscopy has begun to address previously unmet challenges in the study of human disease and is poised to become a pivotal tool in drug discovery.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100209"},"PeriodicalIF":2.7,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143017977","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}
Pub Date : 2025-01-15DOI: 10.1016/j.slasd.2025.100212
Nandini Sharma , Yuka Otsuka , Louis Scampavia , Timothy P. Spicer , Jarrod B. French
Metabolic reprogramming of purine biosynthesis is a hallmark of cancer metabolism and represents a critical vulnerability. The enzyme phosphoribosylformylglycinamidine synthase (PFAS) catalyzes the fourth step in de novo purine biosynthesis and has been demonstrated to be prognostic for survival of liver cancer. Despite the importance of this protein as a drug target, there are no known specific inhibitors of PFAS activity. Here, we describe a new continuous, spectrophotometric assay for the synthase domain of PFAS that is amenable to high-throughput screening (HTS). This mechanism-based fluorescent assay makes use of the acid phosphatase substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). PFAS catalyzes the turnover of DiFMUP with a KM of 108 ± 7 µM. After optimization and miniaturization of the assay for 1,536-well format, we conducted a pilot HTS using the LOPAC1280 library. The assay performed extremely well, with an average Z′ of 0.94 ± 0.02, average signal to noise of 5.01 ± 0.06, excellent inter plate correlation, and a hit rate of 1.18 %. This assay provides a critically needed tool to advance the study of PFAS enzymology and will be foundational for the discovery of small molecule inhibitors both as functional probes and for the basis of new drug development.
{"title":"A high throughput assay for phosphoribosylformylglycinamidine synthase","authors":"Nandini Sharma , Yuka Otsuka , Louis Scampavia , Timothy P. Spicer , Jarrod B. French","doi":"10.1016/j.slasd.2025.100212","DOIUrl":"10.1016/j.slasd.2025.100212","url":null,"abstract":"<div><div>Metabolic reprogramming of purine biosynthesis is a hallmark of cancer metabolism and represents a critical vulnerability. The enzyme phosphoribosylformylglycinamidine synthase (PFAS) catalyzes the fourth step in <em>de novo</em> purine biosynthesis and has been demonstrated to be prognostic for survival of liver cancer. Despite the importance of this protein as a drug target, there are no known specific inhibitors of PFAS activity. Here, we describe a new continuous, spectrophotometric assay for the synthase domain of PFAS that is amenable to high-throughput screening (HTS). This mechanism-based fluorescent assay makes use of the acid phosphatase substrate, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP). PFAS catalyzes the turnover of DiFMUP with a <em>K<sub>M</sub></em> of 108 ± 7 µM. After optimization and miniaturization of the assay for 1,536-well format, we conducted a pilot HTS using the LOPAC<sup>1280</sup> library. The assay performed extremely well, with an average Z′ of 0.94 ± 0.02, average signal to noise of 5.01 ± 0.06, excellent inter plate correlation, and a hit rate of 1.18 %. This assay provides a critically needed tool to advance the study of PFAS enzymology and will be foundational for the discovery of small molecule inhibitors both as functional probes and for the basis of new drug development.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100212"},"PeriodicalIF":2.7,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143017937","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}
Pub Date : 2025-01-15DOI: 10.1016/j.slasd.2025.100211
Abdeldjalil Madani, Nadine Alvarez, Steven Park, Madhuvika Murugan, David S. Perlin
The COVID-19 pandemic has emphasized the necessity for rapid and adaptable drug screening platforms against live pathogenic viruses that require high levels of biosafety containment. Conventional antiviral testing is time-consuming and labor-intensive. Here, we outline the design and validation of a semi-automated drug-screening platform for SARS-CoV-2 that utilizes multiple liquid handlers, a stable A549 cell line expressing ACE2 and TMPRSS2 receptors, and a recombinant SARS-CoV-2 strain harboring the nano-luciferase gene. This platform allows for accelerated low-, mid-, and high-throughput screenings by bypassing the virus inactivation and the staining steps compared to assays utilizing fluorescent reporter viruses or immunofluorescence. First, we demonstrated that the luminescence signal obtained at 24 h post-infection is robust and can be used as a surrogate for fluorescent reporter viruses and immunofluorescence assays that require 48 h incubation post infection. We confirmed the susceptibility of the reporter virus to a panel of reference drugs and validated the luminescence signal in 96- and 384-well plates in accordance with NIH criteria for high-throughput screening. The validation assays showed reproducible results, robust Z factor of ≥0.5, and a coefficient of variation of <20% achieved in both 96 and 384-well plate formats. Lastly, we assessed the assay's performance by screening 240 compounds from the MMV Global Health Library, using the 384-well plate format and remdesivir as a control compound. The single point screening resulted in the identification of 48 hits that inhibited more than 50% of the viral growth. We selected the 15 most active compounds to evaluate their inhibitory concentration and their cytotoxicity, which resulted in the confirmation of the 3 most potent and least toxic compounds that were never reported as antivirals. These results confirm that our platform can be reliably employed for rapid drug screening against SARS-CoV-2 and can be easily adapted to other nano-luciferase reporter viruses.
{"title":"Rapid luminescence-based screening method for SARS- CoV-2 inhibitors discovery","authors":"Abdeldjalil Madani, Nadine Alvarez, Steven Park, Madhuvika Murugan, David S. Perlin","doi":"10.1016/j.slasd.2025.100211","DOIUrl":"10.1016/j.slasd.2025.100211","url":null,"abstract":"<div><div>The COVID-19 pandemic has emphasized the necessity for rapid and adaptable drug screening platforms against live pathogenic viruses that require high levels of biosafety containment. Conventional antiviral testing is time-consuming and labor-intensive. Here, we outline the design and validation of a semi-automated drug-screening platform for SARS-CoV-2 that utilizes multiple liquid handlers, a stable A549 cell line expressing ACE2 and TMPRSS2 receptors, and a recombinant SARS-CoV-2 strain harboring the nano-luciferase gene. This platform allows for accelerated low-, mid-, and high-throughput screenings by bypassing the virus inactivation and the staining steps compared to assays utilizing fluorescent reporter viruses or immunofluorescence. First, we demonstrated that the luminescence signal obtained at 24 h post-infection is robust and can be used as a surrogate for fluorescent reporter viruses and immunofluorescence assays that require 48 h incubation post infection. We confirmed the susceptibility of the reporter virus to a panel of reference drugs and validated the luminescence signal in 96- and 384-well plates in accordance with NIH criteria for high-throughput screening. The validation assays showed reproducible results, robust Z factor of ≥0.5, and a coefficient of variation of <20% achieved in both 96 and 384-well plate formats. Lastly, we assessed the assay's performance by screening 240 compounds from the MMV Global Health Library, using the 384-well plate format and remdesivir as a control compound. The single point screening resulted in the identification of 48 hits that inhibited more than 50% of the viral growth. We selected the 15 most active compounds to evaluate their inhibitory concentration and their cytotoxicity, which resulted in the confirmation of the 3 most potent and least toxic compounds that were never reported as antivirals. These results confirm that our platform can be reliably employed for rapid drug screening against SARS-CoV-2 and can be easily adapted to other nano-luciferase reporter viruses.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100211"},"PeriodicalIF":2.7,"publicationDate":"2025-01-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143017979","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}
Pub Date : 2025-01-11DOI: 10.1016/j.slasd.2025.100210
Francisca Arez , Lena Preiss , Isabella Ramella Gal , Sofia P. Rebelo , Lassina Badolo , Catarina Brito , Thomas Spangenberg , Paula M. Alves
Primary human hepatocytes (PHHs) are the preferred cell source to address liver function. Despite originating from the native tissue, one of the bottlenecks when using primary material is the donor-to-donor variability. Cryopreserved PHHs offer a high number of cells from the same donor and standardization of cell isolation and cryopreservation procedures, mitigating some of the inter-donor variability. Still, PHHs from different commercial sources present variability in vitro in several parameters, including viability post-thawing, plating capacity, aggregation potential and culture longevity. Here we combine stirred-tank culture systems, which allow robust aggregation processes, and co-culture approaches with the HepaRG cell line to generate spheroids from cryopreserved PHHs. By employing small-scale stirred-tank culture systems we could cope with the scarce availability and high cost of primary material. In the optimized co-culture conditions we could generate PHH:HepaRG spheroids from 12 donors acquired from 4 different commercial sources. All PHHs showed similar aggregation profiles, forming small compact heterotypic spheroids as early as 3 days in co-culture and were maintained for at least 5 weeks in culture. The heterotypic spheroids maintained the hepatocyte polarization and identity and showed metabolization capacity for 5 main phase I metabolizing enzymes, namely CYP3A4, CYP2C9, CYP1A2, CYP2D6, and CYP2C8. Moreover, the heterotypic spheroids showed the capacity to metabolize a novel compound under clinical development, showing their potential to be employed in drug discovery applications.
Overall, we present a robust aggregation strategy for cryopreserved PHHs from different suppliers, applicable for pharmacological and toxicological in vitro research.
{"title":"Heterotypic spheroids as a strategy for 3D culture of cryopreserved primary human hepatocytes in stirred-tank systems","authors":"Francisca Arez , Lena Preiss , Isabella Ramella Gal , Sofia P. Rebelo , Lassina Badolo , Catarina Brito , Thomas Spangenberg , Paula M. Alves","doi":"10.1016/j.slasd.2025.100210","DOIUrl":"10.1016/j.slasd.2025.100210","url":null,"abstract":"<div><div>Primary human hepatocytes (PHHs) are the preferred cell source to address liver function. Despite originating from the native tissue, one of the bottlenecks when using primary material is the donor-to-donor variability. Cryopreserved PHHs offer a high number of cells from the same donor and standardization of cell isolation and cryopreservation procedures, mitigating some of the inter-donor variability. Still, PHHs from different commercial sources present variability <em>in vitro</em> in several parameters, including viability post-thawing, plating capacity, aggregation potential and culture longevity. Here we combine stirred-tank culture systems, which allow robust aggregation processes, and co-culture approaches with the HepaRG cell line to generate spheroids from cryopreserved PHHs. By employing small-scale stirred-tank culture systems we could cope with the scarce availability and high cost of primary material. In the optimized co-culture conditions we could generate PHH:HepaRG spheroids from 12 donors acquired from 4 different commercial sources. All PHHs showed similar aggregation profiles, forming small compact heterotypic spheroids as early as 3 days in co-culture and were maintained for at least 5 weeks in culture. The heterotypic spheroids maintained the hepatocyte polarization and identity and showed metabolization capacity for 5 main phase I metabolizing enzymes, namely CYP3A4, CYP2C9, CYP1A2, CYP2D6, and CYP2C8. Moreover, the heterotypic spheroids showed the capacity to metabolize a novel compound under clinical development, showing their potential to be employed in drug discovery applications.</div><div>Overall, we present a robust aggregation strategy for cryopreserved PHHs from different suppliers, applicable for pharmacological and toxicological in vitro research.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100210"},"PeriodicalIF":2.7,"publicationDate":"2025-01-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142980832","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}
Pub Date : 2025-01-06DOI: 10.1016/j.slasd.2025.100208
Marcus Y. Chin , David A. Joy , Madhuja Samaddar, Anil Rana, Johann Chow, Takashi Miyamoto, Meredith Calvert
Mitochondria undergo dynamic morphological changes depending on cellular cues, stress, genetic factors, or disease. The structural complexity and disease-relevance of mitochondria have stimulated efforts to generate image analysis tools for describing mitochondrial morphology for therapeutic development. Using high-content analysis, we measured multiple morphological parameters and employed unbiased feature clustering to identify the most robust pair of texture metrics that described mitochondrial state. Here, we introduce a novel image analysis pipeline to enable rapid and accurate profiling of mitochondrial morphology in various cell types and pharmacological perturbations. We applied a high-content adapted implementation of our tool, MitoProfilerHC, to quantify mitochondrial morphology changes in i) a mammalian cell dose response study and ii) compartment-specific drug effects in primary neurons. Next, we expanded the usability of our pipeline by using napari, a Python-powered image analysis tool, to build an open-source version of MitoProfiler and validated its performance and applicability. In conclusion, we introduce MitoProfiler as both a high-content-based and an open-source method to accurately quantify mitochondrial morphology in cells, which we anticipate to greatly facilitate mechanistic discoveries in mitochondrial biology and disease.
{"title":"Novel high-content and open-source image analysis tools for profiling mitochondrial morphology in neurological cell models","authors":"Marcus Y. Chin , David A. Joy , Madhuja Samaddar, Anil Rana, Johann Chow, Takashi Miyamoto, Meredith Calvert","doi":"10.1016/j.slasd.2025.100208","DOIUrl":"10.1016/j.slasd.2025.100208","url":null,"abstract":"<div><div>Mitochondria undergo dynamic morphological changes depending on cellular cues, stress, genetic factors, or disease. The structural complexity and disease-relevance of mitochondria have stimulated efforts to generate image analysis tools for describing mitochondrial morphology for therapeutic development. Using high-content analysis, we measured multiple morphological parameters and employed unbiased feature clustering to identify the most robust pair of texture metrics that described mitochondrial state. Here, we introduce a novel image analysis pipeline to enable rapid and accurate profiling of mitochondrial morphology in various cell types and pharmacological perturbations. We applied a high-content adapted implementation of our tool, MitoProfilerHC, to quantify mitochondrial morphology changes in i) a mammalian cell dose response study and ii) compartment-specific drug effects in primary neurons. Next, we expanded the usability of our pipeline by using napari, a Python-powered image analysis tool, to build an open-source version of MitoProfiler and validated its performance and applicability. In conclusion, we introduce MitoProfiler as both a high-content-based and an open-source method to accurately quantify mitochondrial morphology in cells, which we anticipate to greatly facilitate mechanistic discoveries in mitochondrial biology and disease.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"31 ","pages":"Article 100208"},"PeriodicalIF":2.7,"publicationDate":"2025-01-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142960156","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}
Pub Date : 2025-01-01DOI: 10.1016/j.slasd.2024.100207
David A. Close , V. Blair Journigan , Paul A. Johnston
Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca2+ mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca2+ mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.2 % was set. The resting baseline relative fluorescent unit (RFUs) signals of the TRPM8 Ca2+ mobilization assay exhibited substantial well-to-well variability, even though such differences were small relative to maximal agonist induced responses. Maximum RFU, cumulative RFU sum, or area under the curve values were extracted from Ca2+ mobilization kinetic data to plot curves and calculate EC50 and IC50 values. Fold over baseline (FOB) ratio data processing eliminated well-to-well differences in resting baseline signals, reduced error bars, improved curve fits and reduced 95 % confidence interval EC50 and IC50 ranges. FOB ratio data processing decreased variability and improved the precision of repeat measurements in single experimental sessions thereby reducing the minimum threshold difference in EC50 or IC50 values required to distinguish compound potencies. EC50 and IC50 values of TRPM8 agonists and antagonists determined in single experiments were strongly aligned to those from multiple independent experiments. Benchmark TRPM8, TRPV1, and TRPA1 EC50 and IC50 values were within the ranges previously reported for agonist and antagonist standards. The improved precision and accuracy of the TRP Ca2+ mobilization assays afforded by FOB ratio data processing enhances their utility for investigating structure activity relationships.
{"title":"Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1","authors":"David A. Close , V. Blair Journigan , Paul A. Johnston","doi":"10.1016/j.slasd.2024.100207","DOIUrl":"10.1016/j.slasd.2024.100207","url":null,"abstract":"<div><div>Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca<sup>2+</sup> mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca<sup>2+</sup> mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.2 % was set. The resting baseline relative fluorescent unit (RFUs) signals of the TRPM8 Ca<sup>2+</sup> mobilization assay exhibited substantial well-to-well variability, even though such differences were small relative to maximal agonist induced responses. Maximum RFU, cumulative RFU sum, or area under the curve values were extracted from Ca<sup>2+</sup> mobilization kinetic data to plot curves and calculate EC<sub>50</sub> and IC<sub>50</sub> values. Fold over baseline (FOB) ratio data processing eliminated well-to-well differences in resting baseline signals, reduced error bars, improved curve fits and reduced 95 % confidence interval EC<sub>50</sub> and IC<sub>50</sub> ranges. FOB ratio data processing decreased variability and improved the precision of repeat measurements in single experimental sessions thereby reducing the minimum threshold difference in EC<sub>50</sub> or IC<sub>50</sub> values required to distinguish compound potencies. EC<sub>50</sub> and IC<sub>50</sub> values of TRPM8 agonists and antagonists determined in single experiments were strongly aligned to those from multiple independent experiments. Benchmark TRPM8, TRPV1, and TRPA1 EC<sub>50</sub> and IC<sub>50</sub> values were within the ranges previously reported for agonist and antagonist standards. The improved precision and accuracy of the TRP Ca2+ mobilization assays afforded by FOB ratio data processing enhances their utility for investigating structure activity relationships.</div></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"30 ","pages":"Article 100207"},"PeriodicalIF":2.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142924269","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}
Pub Date : 2025-01-01DOI: 10.1016/j.slasd.2024.100200
Namrata Jayanth , Gurvan Mahé , Matthew Campbell , Mike Lipkin , Shushant Jain , Rhea van de Bospoort , Jennifer Thornton , Brad Margus , David F. Fischer
Ataxia Telangiectasia (A-T) is a rare, autosomal recessive genetic disorder characterized by a variety of symptoms, including progressive neurodegeneration, telangiectasia, immunodeficiency, and an increased susceptibility to cancer. It is caused by bi-allelic mutations impacting a gene encoding a serine/threonine kinase ATM (Ataxia Telangiectasia Mutated), which plays a crucial role in DNA repair and maintenance of genomic stability. The disorder primarily affects the nervous system, leading to a range of neurological issues, including cerebellar ataxia. The cause of neurodegeneration due to mutations in ATM is still an area of investigation, and currently there is no known treatment to slow down or stop the progression of the neurological problems.
In this collaboration of the A-T Children's Project (ATCP) with Charles River Discovery, we successfully developed a high-throughput assay using induced pluripotent stem cells (iPSC) from A-T donors to measure DNA damage response (DDR). By measuring the changes in levels of activated phosphorylated CHK2 (p-CHK2), which is a downstream signaling event of ATM, we were able to identify compounds that restore this response in the DDR pathway in A-T derived patient cells. Over 6,000 compounds from small molecule drug repurposing libraries were subsequently screened in the assay developed, leading to identification of several promising in vitro hits.
Using the assay developed and the identified hits opens avenues to investigate potential therapeutics for A-T.
共济失调毛细血管扩张症（a - t）是一种罕见的常染色体隐性遗传病，其特征是多种症状，包括进行性神经变性、毛细血管扩张、免疫缺陷和对癌症的易感性增加。它是由双等位基因突变影响编码丝氨酸/苏氨酸激酶ATM（共济失调毛细血管扩张突变）的基因引起的，该基因在DNA修复和基因组稳定性的维持中起着至关重要的作用。这种疾病主要影响神经系统，导致一系列神经问题，包括小脑性共济失调。由ATM突变引起的神经退行性变的原因仍是一个研究领域，目前还没有已知的治疗方法来减缓或阻止神经问题的进展。在a -t儿童项目（ATCP）与Charles River Discovery的合作中，我们成功开发了一种使用来自a -t供体的诱导多能干细胞（iPSC）来测量DNA损伤反应（DDR）的高通量测定方法。通过测量活化磷酸化CHK2 （p-CHK2）水平的变化，这是ATM的下游信号事件，我们能够识别在a - t衍生的患者细胞中恢复DDR途径中这种反应的化合物。随后，从小分子药物再利用文库中筛选了6000多种化合物，并确定了几种有前景的体外命中。使用开发的检测方法和确定的命中点为研究A-T的潜在治疗方法开辟了道路。
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Pub Date : 2025-01-01DOI: 10.1016/j.slasd.2024.100201
David A. Close , Paul A. Johnston
Patient derived tumor organoid (PDTO) models retain the structural, morphological, genetic, and clonal heterogeneity of the original tumors. The ability to efficiently generate, expand, and biobank PDTOs has the potential to make the clinical diversity of cancer accessible for personalized medicine assay guided therapeutic drug selection and drug discovery. We describe the miniaturization and growth in 96- and 384-well formats of a single non-tumor liver and two Hepatocellular carcinoma (HCC) organoids derived from cryopreserved PDTO cells and the application of high content imaging (HCI) to characterize the models and enhance drug sensitivity testing. Non-invasive sequentially acquired transmitted light images showed that seeding cryopreserved cells from non-tumoral and HCC PDTOs into 96- or 384-well plates in reduced growth factor Matrigel (rgf-MG) that were fed with growth medium every 3 days supported organoid growth up to 15 days. The number and sizes of organoids increased with longer times in culture. HCC PDTO's had more heterogeneous morphologies than non-tumor organoids with respect to size, shape, and optical density. Organoids cultured in rgf-MG could be stained in situ with HCI reagents without mechanical, chemical or enzymatic disruption of the hydrogel matrices and quantitative data extracted by image analysis. Hoechst and live/dead reagents provided organoid numbers and viability comparisons. HCC PDTO's stained with phalloidin or immuno-stained with α-tubulin antibodies revealed F-actin and microtubule cytoskeleton organization. HCC PDTO's stained with antibodies to signaling pathway proteins and their phosphorylation status allowed comparisons of relative expression levels and inference of pathway activation. Images of HCC PDTO's exposed to ellipticine showed that drugs penetrate Matrigel hydrogels and accumulate in organoid cells. 9-day 384-well HCC organoid cultures exhibited robust and reproducible growth signals suitable for cancer drug testing. Complimenting cell viability readouts with multiple HCI parameters including morphological features and dead cell staining improved the analysis of drug impact and enhanced the value that could be extracted from these more physiologically relevant three-dimensional HCC organoid cultures.
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