Pub Date : 2024-05-24DOI: 10.1016/j.slasd.2024.100162
Lillie E. Bell , Catherine Bardelle , Martin J Packer , Johanna Kastl , Geoffrey A. Holdgate , Gareth Davies
Targeted protein degradation is an important mechanism carried out by the cellular machinery, one that is gaining momentum as an exploitable strategy for the development of drug-like compounds. Molecules which are able to induce proximity between elusive therapeutic targets of interest and E3 ligases which subsequently leads to proteasomal degradation of the target are beginning to decrease the percentage of the human proteome described as undruggable. Therefore, having the ability to screen for, and understand the mechanism of, such molecules is becoming an increasingly attractive scientific focus. We have established a number of cascade experiments including cell-based assays and orthogonal triage steps to provide annotation to the selectivity and mechanism of action for compounds identified as putative degraders from a primary high throughput screen against a high value oncology target. We will describe our current position, using PROTACs as proof-of-concept, on the analysis of these novel outputs and highlight challenges encountered.
{"title":"Characterisation of high throughput screening outputs for small molecule degrader discovery","authors":"Lillie E. Bell , Catherine Bardelle , Martin J Packer , Johanna Kastl , Geoffrey A. Holdgate , Gareth Davies","doi":"10.1016/j.slasd.2024.100162","DOIUrl":"10.1016/j.slasd.2024.100162","url":null,"abstract":"<div><p>Targeted protein degradation is an important mechanism carried out by the cellular machinery, one that is gaining momentum as an exploitable strategy for the development of drug-like compounds. Molecules which are able to induce proximity between elusive therapeutic targets of interest and E3 ligases which subsequently leads to proteasomal degradation of the target are beginning to decrease the percentage of the human proteome described as undruggable. Therefore, having the ability to screen for, and understand the mechanism of, such molecules is becoming an increasingly attractive scientific focus. We have established a number of cascade experiments including cell-based assays and orthogonal triage steps to provide annotation to the selectivity and mechanism of action for compounds identified as putative degraders from a primary high throughput screen against a high value oncology target. We will describe our current position, using PROTACs as proof-of-concept, on the analysis of these novel outputs and highlight challenges encountered.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 5","pages":"Article 100162"},"PeriodicalIF":3.1,"publicationDate":"2024-05-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000248/pdfft?md5=dc288a87ff98b6fd26cc25724269aadb&pid=1-s2.0-S2472555224000248-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141140527","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 : 2024-05-07DOI: 10.1016/j.slasd.2024.100159
Christopher Fotsch , Debaleena Basu , Ryan Case , Qing Chen , Pratibha C. Koneru , Mei-Chu Lo , Rachel Ngo , Pooja Sharma , Amit Vaish , Xiang Yi , Stephan G. Zech , Peter Hodder
To confirm target engagement of hits from our high-throughput screening efforts, we ran biophysical assays on several hundreds of hits from 15 different high-throughput screening campaigns. Analyzing the biophysical assay results from these screening campaigns led us to conclude that we could be more strategic in our biophysical analysis of hits by first confirming activity in a thermal shift assay (TSA) and then confirming activity in either a surface plasmon resonance (SPR) assay or a temperature-related intensity change (TRIC) assay. To understand how this new workflow shapes the quality of the final hits, we compared TSA/SPR or TSA/TRIC confirmed and unconfirmed hits to one another using four measures of compound quality: quantitative estimate of drug-likeness (QED), Pan-Assay Interference Compounds (PAINS), promiscuity, and aqueous solubility. In general, we found that the biophysically confirmed hits performed better in the compound quality metrics than the unconfirmed hits, demonstrating that our workflow not only confirmed target engagement of the hits but also enriched for higher quality hits.
{"title":"Creating a more strategic small molecule biophysical hit characterization workflow","authors":"Christopher Fotsch , Debaleena Basu , Ryan Case , Qing Chen , Pratibha C. Koneru , Mei-Chu Lo , Rachel Ngo , Pooja Sharma , Amit Vaish , Xiang Yi , Stephan G. Zech , Peter Hodder","doi":"10.1016/j.slasd.2024.100159","DOIUrl":"10.1016/j.slasd.2024.100159","url":null,"abstract":"<div><p>To confirm target engagement of hits from our high-throughput screening efforts, we ran biophysical assays on several hundreds of hits from 15 different high-throughput screening campaigns. Analyzing the biophysical assay results from these screening campaigns led us to conclude that we could be more strategic in our biophysical analysis of hits by first confirming activity in a thermal shift assay (TSA) and then confirming activity in either a surface plasmon resonance (SPR) assay or a temperature-related intensity change (TRIC) assay. To understand how this new workflow shapes the quality of the final hits, we compared TSA/SPR or TSA/TRIC confirmed and unconfirmed hits to one another using four measures of compound quality: quantitative estimate of drug-likeness (QED), Pan-Assay Interference Compounds (PAINS), promiscuity, and aqueous solubility. In general, we found that the biophysically confirmed hits performed better in the compound quality metrics than the unconfirmed hits, demonstrating that our workflow not only confirmed target engagement of the hits but also enriched for higher quality hits.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 4","pages":"Article 100159"},"PeriodicalIF":3.1,"publicationDate":"2024-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000212/pdfft?md5=389ec9b1863fb13b54ef5a3823355f2d&pid=1-s2.0-S2472555224000212-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140900579","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 : 2024-05-01DOI: 10.1016/j.slasd.2024.100158
Taís Bacelar Sacramento de Araújo , Raphael Luís Rocha Nogueira , Leonardo de Oliveira Siquara da Rocha , Iasmin Nogueira Bastos , Rosane Borges Dias , Bruno Solano De Freitas Souza , Daniel William Lambert , Ricardo D. Coletta , Viviane Aline Oliveira Silva , Clarissa A. Gurgel Rocha
3D in vitro systems offer advantages over the shortcomings of two-dimensional models by simulating the morphological and functional features of in vivo-like environments, such as cell-cell and cell-extracellular matrix interactions, as well as the co-culture of different cell types. Nevertheless, these systems present technical challenges that limit their potential in cancer research requiring cell line- and culture-dependent standardization. This protocol details the use of a magnetic 3D bioprinting method and other associated techniques (cytotoxicity assay and histological analysis) using oral squamous cell carcinoma cell line, HSC3, which offer advantages compared to existing widely used approaches. This protocol is particularly timely, as it validates magnetic bioprinting as a method for the rapid deployment of 3D cultures as a tool for compound screening and development of heterotypic cultures such as co-culture of oral squamous cell carcinoma cells with cancer-associated fibroblasts (HSC3/CAFs).
三维体外系统通过模拟类似活体环境的形态和功能特征,如细胞-细胞和细胞-细胞外基质的相互作用,以及不同类型细胞的共培养,克服了二维模型的缺点。然而,这些系统在技术上存在挑战,限制了它们在癌症研究中的潜力,因为癌症研究需要依赖细胞系和培养的标准化。本方案详细介绍了利用口腔鳞状细胞癌细胞系 HSC3 使用磁性 3D 生物打印方法和其他相关技术(细胞毒性检测和组织学分析)的情况,与现有的广泛使用的方法相比,这种方法具有优势。该方案非常及时,因为它验证了磁性三维生物打印是一种快速部署三维培养物的方法,可作为化合物筛选和开发异型培养物(如口腔鳞状细胞癌细胞与癌症相关成纤维细胞(HSC3/CAFs)的共培养)的工具。
{"title":"Enhancing scaffold-free spheroid models: 3D cell bioprinting method for metastatic HSC3–Oral squamous carcinoma cell line","authors":"Taís Bacelar Sacramento de Araújo , Raphael Luís Rocha Nogueira , Leonardo de Oliveira Siquara da Rocha , Iasmin Nogueira Bastos , Rosane Borges Dias , Bruno Solano De Freitas Souza , Daniel William Lambert , Ricardo D. Coletta , Viviane Aline Oliveira Silva , Clarissa A. Gurgel Rocha","doi":"10.1016/j.slasd.2024.100158","DOIUrl":"https://doi.org/10.1016/j.slasd.2024.100158","url":null,"abstract":"<div><p>3D <em>in vitro</em> systems offer advantages over the shortcomings of two-dimensional models by simulating the morphological and functional features of <em>in vivo</em>-like environments, such as cell-cell and cell-extracellular matrix interactions, as well as the co-culture of different cell types. Nevertheless, these systems present technical challenges that limit their potential in cancer research requiring cell line- and culture-dependent standardization. This protocol details the use of a magnetic 3D bioprinting method and other associated techniques (cytotoxicity assay and histological analysis) using oral squamous cell carcinoma cell line, HSC3, which offer advantages compared to existing widely used approaches. This protocol is particularly timely, as it validates magnetic bioprinting as a method for the rapid deployment of 3D cultures as a tool for compound screening and development of heterotypic cultures such as co-culture of oral squamous cell carcinoma cells with cancer-associated fibroblasts (HSC3/CAFs).</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 4","pages":"Article 100158"},"PeriodicalIF":3.1,"publicationDate":"2024-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000200/pdfft?md5=65af641e1cc59f0ba7520fa27146fec1&pid=1-s2.0-S2472555224000200-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140924481","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 : 2024-04-18DOI: 10.1016/j.slasd.2024.100156
Alice Lanne , Catherine Bardelle , Gareth Davies , Antonia Turberville , Hannah Semple , Rachel Moore , Geoffrey A. Holdgate
Fluorescent probe modulation assays are a widely used approach to monitor displacement or stabilisation of fluorescently labelled tool ligands by test compounds. These assays allow an optical read-out of probe-receptor binding and can be used to detect compounds that compete with the labelled ligand, either directly or indirectly. Probes for both orthosteric and allosteric sites are often employed. The method can also be used to identify test compounds that may stabilise the ternary complex, offering an opportunity to discover novel molecular glues. The utility of these fluorescence-based assays within high-throughput screening has been facilitated by the use of streptavidin labelled terbium as a donor and access to a range of different acceptor fluorophores. During 2023, the High-throughput Screening group at AstraZeneca carried out 8 high-throughput screens using these approaches. In this manuscript we will present the types of assays used, an overview of the timelines for assay development and screening, the application of orthogonal artefact methods to aid hit finding and the results of the screens in terms of hit rate and the number of compounds identified with IC50 values of better than 30 µM. Learning across the development, execution and analysis of these screens will be presented.
{"title":"POLARISED views and FRETting about probe modulation assays: Learning from High Throughput Screening","authors":"Alice Lanne , Catherine Bardelle , Gareth Davies , Antonia Turberville , Hannah Semple , Rachel Moore , Geoffrey A. Holdgate","doi":"10.1016/j.slasd.2024.100156","DOIUrl":"https://doi.org/10.1016/j.slasd.2024.100156","url":null,"abstract":"<div><p>Fluorescent probe modulation assays are a widely used approach to monitor displacement or stabilisation of fluorescently labelled tool ligands by test compounds. These assays allow an optical read-out of probe-receptor binding and can be used to detect compounds that compete with the labelled ligand, either directly or indirectly. Probes for both orthosteric and allosteric sites are often employed. The method can also be used to identify test compounds that may stabilise the ternary complex, offering an opportunity to discover novel molecular glues. The utility of these fluorescence-based assays within high-throughput screening has been facilitated by the use of streptavidin labelled terbium as a donor and access to a range of different acceptor fluorophores. During 2023, the High-throughput Screening group at AstraZeneca carried out 8 high-throughput screens using these approaches. In this manuscript we will present the types of assays used, an overview of the timelines for assay development and screening, the application of orthogonal artefact methods to aid hit finding and the results of the screens in terms of hit rate and the number of compounds identified with IC<sub>50</sub> values of better than 30 µM. Learning across the development, execution and analysis of these screens will be presented.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 4","pages":"Article 100156"},"PeriodicalIF":3.1,"publicationDate":"2024-04-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000182/pdfft?md5=31508b5b2d14cbf4e0c02d5d7e69cecf&pid=1-s2.0-S2472555224000182-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140645364","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 : 2024-04-01DOI: 10.1016/j.slasd.2024.100154
Inês C. Ferreira , Estefania Torrejón , Bernardo Abecasis , Bruno M. Alexandre , Ricardo A. Gomes , Chris Verslype , Jos van Pelt , Ana Barbas , Daniel Simão , Tiago M. Bandeiras , Alessio Bortoluzzi , Sofia P. Rebelo
Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale. Among the potential sorafenib targets, we identified aldehyde dehydrogenase 2 (ALDH2), an enzyme that plays a major role in alcohol metabolism. We validated the interaction of sorafenib with ALDH2 by orthogonal methods using pure recombinant protein, proving that this interaction is not mediated by other cellular components. Moreover, we showed that sorafenib inhibits ALDH2 activity, supporting a functional role for this interaction. Finally, we were able to demonstrate that both ALDH2 protein expression and activity were reduced in sorafenib-resistant cells compared to the parental cell line. Overall, our study allowed the identification of ALDH2 as a novel sorafenib target and sheds light on its potential role in both hepatocellular carcinoma and sorafenib resistance condition.
{"title":"Aldehyde Dehydrogenase 2 (ALDH2): A novel sorafenib target in hepatocellular carcinoma unraveled by the proteome-wide cellular thermal shift assay","authors":"Inês C. Ferreira , Estefania Torrejón , Bernardo Abecasis , Bruno M. Alexandre , Ricardo A. Gomes , Chris Verslype , Jos van Pelt , Ana Barbas , Daniel Simão , Tiago M. Bandeiras , Alessio Bortoluzzi , Sofia P. Rebelo","doi":"10.1016/j.slasd.2024.100154","DOIUrl":"10.1016/j.slasd.2024.100154","url":null,"abstract":"<div><p>Sorafenib is a multikinase inhibitor indicated for first-line treatment of unresectable hepatocellular carcinoma. Despite its widespread use in the clinic, the existing knowledge of sorafenib mode-of-action remains incomplete. To build upon the current understanding, we used the Cellular Thermal Shift Assay (CETSA) coupled to Mass Spectrometry (CETSA-MS) to monitor compound binding to its target proteins in the cellular context on a proteome-wide scale. Among the potential sorafenib targets, we identified aldehyde dehydrogenase 2 (ALDH2), an enzyme that plays a major role in alcohol metabolism. We validated the interaction of sorafenib with ALDH2 by orthogonal methods using pure recombinant protein, proving that this interaction is not mediated by other cellular components. Moreover, we showed that sorafenib inhibits ALDH2 activity, supporting a functional role for this interaction. Finally, we were able to demonstrate that both ALDH2 protein expression and activity were reduced in sorafenib-resistant cells compared to the parental cell line. Overall, our study allowed the identification of ALDH2 as a novel sorafenib target and sheds light on its potential role in both hepatocellular carcinoma and sorafenib resistance condition.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 3","pages":"Article 100154"},"PeriodicalIF":3.1,"publicationDate":"2024-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000169/pdfft?md5=e39307bdcc31a11896db9724bd824787&pid=1-s2.0-S2472555224000169-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140195277","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 : 2024-03-20DOI: 10.1016/j.slasd.2024.100153
Wenwen Zhou , Baoqing You , Xiaomeng Zhao , Shuyi Si , Yan Li , Jing Zhang
Cathepsin L (CTSL), a lysosomal cysteine proteinase, is primarily dedicated to the metabolic turnover of intracellular proteins. It is involved in various physiological processes and contributes to pathological conditions such as viral infection, tumor invasion and metastasis, inflammatory status, atherosclerosis, renal disease, diabetes, bone diseases, and other ailments. The coronavirus disease 2019 (COVID-19), with its rapid global spread and significant mortality, has been a worldwide epidemic since the late 2019s. Notably, CTSL plays a role in the processing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, providing a potential avenue to block coronavirus host cell entry and thereby inhibit SARS-CoV-2 infection in humans. In this study, we have developed a novel method using fluorescence polarization (FP) for screening CTSL inhibitors in a high-throughput format. The optimized assay demonstrated its appropriateness for high-throughput screening (HTS) with a Z-factor of 0.9 in a 96-well format. Additionally, the IC50 of the known inhibitor, Z-Phe-Tyr-CHO, was determined to be 188.50 ± 46.88 nM. Upon screening over 2000 small molecules, we identified, for the first time, the anti-CTSL properties of a benzothiazoles derivative named IMB 8015. This work presents a novel high-throughput approach and its application in discovering and evaluating CTSL inhibitors.
{"title":"Establishment, optimization and validation of a fluorescence polarization-based high-throughput screening assay targeting cathepsin L inhibitors","authors":"Wenwen Zhou , Baoqing You , Xiaomeng Zhao , Shuyi Si , Yan Li , Jing Zhang","doi":"10.1016/j.slasd.2024.100153","DOIUrl":"10.1016/j.slasd.2024.100153","url":null,"abstract":"<div><p>Cathepsin L (CTSL), a lysosomal cysteine proteinase, is primarily dedicated to the metabolic turnover of intracellular proteins. It is involved in various physiological processes and contributes to pathological conditions such as viral infection, tumor invasion and metastasis, inflammatory status, atherosclerosis, renal disease, diabetes, bone diseases, and other ailments. The coronavirus disease 2019 (COVID-19), with its rapid global spread and significant mortality, has been a worldwide epidemic since the late 2019s. Notably, CTSL plays a role in the processing of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, providing a potential avenue to block coronavirus host cell entry and thereby inhibit SARS-CoV-2 infection in humans. In this study, we have developed a novel method using fluorescence polarization (FP) for screening CTSL inhibitors in a high-throughput format. The optimized assay demonstrated its appropriateness for high-throughput screening (HTS) with a Z-factor of 0.9 in a 96-well format. Additionally, the IC<sub>50</sub> of the known inhibitor, Z-Phe-Tyr-CHO, was determined to be 188.50 ± 46.88 nM. Upon screening over 2000 small molecules, we identified, for the first time, the anti-CTSL properties of a benzothiazoles derivative named IMB 8015. This work presents a novel high-throughput approach and its application in discovering and evaluating CTSL inhibitors.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 3","pages":"Article 100153"},"PeriodicalIF":3.1,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000157/pdfft?md5=531dea274a50b4826ec3758fe007d67d&pid=1-s2.0-S2472555224000157-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140190478","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 : 2024-03-20DOI: 10.1016/j.slasd.2024.100155
Wenyu Wang , Jing Tang , Andrea Zaliani
In June 2022, EU-OS came to the decision to make public a solubility data set of 100+K compounds obtained from several of the EU-OS proprietary screening compound collections. Leveraging on the interest of SLAS for screening scientific development it was decided to launch a joint EUOS-SLAS competition within the chemoinformatics and machine learning (ML) communities. The competition was open to real world computation experts, for the best, most predictive, classification model of compound solubility. The aim of the competition was multiple: from a practical side, the winning model should then serve as a cornerstone for future solubility predictions having used the largest training set so far publicly available. From a higher project perspective, the intent was to focus the energies and experiences, even if professionally not precisely coming from Pharma R&D; to address the issue of how to predict compound solubility. Here we report how the competition was ideated and the practical aspects of conducting it within the Kaggle framework, leveraging of the versatility and the open-source nature of this data science platform. Consideration on results and challenges encountered have been also examined.
{"title":"Outline and background for the EU-OS solubility prediction challenge","authors":"Wenyu Wang , Jing Tang , Andrea Zaliani","doi":"10.1016/j.slasd.2024.100155","DOIUrl":"10.1016/j.slasd.2024.100155","url":null,"abstract":"<div><p>In June 2022, EU-OS came to the decision to make public a solubility data set of 100+K compounds obtained from several of the EU-OS proprietary screening compound collections. Leveraging on the interest of SLAS for screening scientific development it was decided to launch a joint EUOS-SLAS competition within the chemoinformatics and machine learning (ML) communities. The competition was open to real world computation experts, for the best, most predictive, classification model of compound solubility. The aim of the competition was multiple: from a practical side, the winning model should then serve as a cornerstone for future solubility predictions having used the largest training set so far publicly available. From a higher project perspective, the intent was to focus the energies and experiences, even if professionally not precisely coming from Pharma R&D; to address the issue of how to predict compound solubility. Here we report how the competition was ideated and the practical aspects of conducting it within the Kaggle framework, leveraging of the versatility and the open-source nature of this data science platform. Consideration on results and challenges encountered have been also examined.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 4","pages":"Article 100155"},"PeriodicalIF":3.1,"publicationDate":"2024-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000170/pdfft?md5=31adf40156a4682f7a09408654d5d462&pid=1-s2.0-S2472555224000170-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140190479","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 : 2024-03-06DOI: 10.1016/j.slasd.2024.100149
Jose Brea , Maria J. Varela , Geert A. Daudey , Maria I. Loza
The purpose of the protocol reported in this work is the solubility profiling of large chemical libraries using nephelometry. This technique allows the qualitative classification of compounds as highly, moderately, or poorly water-soluble. The described methodology is not intended to yield quantitative solubility values of the studied compounds but can be used as a primary solubility assessment of large chemical libraries, to guide hit prioritization after High Throughput Screening (HTS) campaigns.
{"title":"High-throughput nephelometry methodology for qualitative determination of aqueous solubility of chemical libraries","authors":"Jose Brea , Maria J. Varela , Geert A. Daudey , Maria I. Loza","doi":"10.1016/j.slasd.2024.100149","DOIUrl":"10.1016/j.slasd.2024.100149","url":null,"abstract":"<div><p>The purpose of the protocol reported in this work is the solubility profiling of large chemical libraries using nephelometry. This technique allows the qualitative classification of compounds as highly, moderately, or poorly water-soluble. The described methodology is not intended to yield quantitative solubility values of the studied compounds but can be used as a primary solubility assessment of large chemical libraries, to guide hit prioritization after High Throughput Screening (HTS) campaigns.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 3","pages":"Article 100149"},"PeriodicalIF":3.1,"publicationDate":"2024-03-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S247255522400011X/pdfft?md5=a9b754a03190a54dd2fb6bb4cd348956&pid=1-s2.0-S247255522400011X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140084712","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 : 2024-03-02DOI: 10.1016/j.slasd.2024.100148
Emery Smith , Louise Dickson , Philip Pickford , Anna Rowland , Justin Shumate , Katherine Perez , Louis Scampavia , Derek Hernandez , Timothy P. Spicer
Fluorescence-based potassium channel assays are typically run on expensive, hard to obtain, fluorescence imaging kinetic plate readers that are uncommon in most laboratories. Here we describe the use of the Brilliant Thallium Snapshot assay to conduct an endpoint potassium channel assay, so that it can be used across multiple plate reader platforms that are more common in many labs. These methods will allow users to identify modulators of potassium channels. For this work, we have taken a kinetic mode Molecular Devices FLIPR based protocol and adapted it to be utilized on endpoint plate readers, such as the BMG Labtech PHERAstar, to identify activators of GIRK channels in CHO cells. We demonstrate that both plate readers are functionally competent at generating excellent Z’ values which makes them ideally suited to finding corollary hits from the Sigma LOPAC 1,280 screening collection. Importantly, this assay has also been validated using a high content reader, demonstrating the possibility of spatially resolving signals from individual cells within a mixed cell population. The compendium of these results shows the flexibility, accessibility and functionality of endpoint-compatible potassium channel assay readouts on more common plate readers.
{"title":"Protocol for kinetic mode potassium channel assays on common plate readers and microscopes","authors":"Emery Smith , Louise Dickson , Philip Pickford , Anna Rowland , Justin Shumate , Katherine Perez , Louis Scampavia , Derek Hernandez , Timothy P. Spicer","doi":"10.1016/j.slasd.2024.100148","DOIUrl":"https://doi.org/10.1016/j.slasd.2024.100148","url":null,"abstract":"<div><p>Fluorescence-based potassium channel assays are typically run on expensive, hard to obtain, fluorescence imaging kinetic plate readers that are uncommon in most laboratories. Here we describe the use of the Brilliant Thallium Snapshot assay to conduct an endpoint potassium channel assay, so that it can be used across multiple plate reader platforms that are more common in many labs. These methods will allow users to identify modulators of potassium channels. For this work, we have taken a kinetic mode Molecular Devices FLIPR based protocol and adapted it to be utilized on endpoint plate readers, such as the BMG Labtech PHERAstar, to identify activators of GIRK channels in CHO cells. We demonstrate that both plate readers are functionally competent at generating excellent Z’ values which makes them ideally suited to finding corollary hits from the Sigma LOPAC 1,280 screening collection. Importantly, this assay has also been validated using a high content reader, demonstrating the possibility of spatially resolving signals from individual cells within a mixed cell population. The compendium of these results shows the flexibility, accessibility and functionality of endpoint-compatible potassium channel assay readouts on more common plate readers.</p></div>","PeriodicalId":21764,"journal":{"name":"SLAS Discovery","volume":"29 3","pages":"Article 100148"},"PeriodicalIF":3.1,"publicationDate":"2024-03-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2472555224000108/pdfft?md5=fb5fd387899cb3c039a7cbf52f959893&pid=1-s2.0-S2472555224000108-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140052681","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 : 2024-03-01DOI: 10.1016/j.slasd.2023.12.009
Lamiaa Bahnassawy , Nathalie Nicolaisen , Christopher Untucht , Benjamin Mielich-Süss , Lydia Reinhardt , Janina S. Ried , Martina P. Morawe , Daniela Geist , Anja Finck , Elke Käfer , Jürgen Korffmann , Matthew Townsend , Brinda Ravikumar , Viktor Lakics , Miroslav Cik , Peter Reinhardt
Aberrant protein aggregation is a pathological cellular hallmark of many neurodegenerative diseases, such as Alzheimer's disease (AD) and frontotemporal dementia (FTD), where the tau protein is aggregating, forming neurofibrillary tangles (NFTs), and propagating from neuron to neuron. These processes have been linked to disease progression and a decline in cognitive function. Various therapeutic approaches aim at the prevention or reduction of tau aggregates in neurons. Human induced pluripotent stem cells (hiPSCs) are a very valuable tool in neuroscience discovery, as they offer access to potentially unlimited amounts of cell types that are affected in disease, including cortical neurons, for in vitro studies. We have generated an in vitro model for tau aggregation that uses hiPSC – derived neurons expressing an aggregation prone, fluorescently tagged version of the human tau protein after lentiviral transduction. Upon addition of tau seeds in the form of recombinant sonicated paired helical filaments (sPHFs), the neurons show robust, disease-like aggregation of the tau protein. The model was developed as a plate-based high content screening assay coupled with an image analysis algorithm to evaluate the impact of small molecules or genetic perturbations on tau. We show that the assay can be used to evaluate small molecules or screen targeted compound libraries. Using siRNA-based gene knockdown, genes of interest can be evaluated, and we could show that a targeted gene library can be screened, by screening nearly 100 deubiquitinating enzymes (DUBs) in that assay. The assay uses an imaging-based readout, a relatively short timeline, quantifies the extent of tau aggregation, and also allows the assessment of cell viability. Furthermore, it can be easily adapted to different hiPSC lines or neuronal subtypes. Taken together, this complex and highly relevant approach can be routinely applied on a weekly basis in the screening funnels of several projects and generates data with a turnaround time of approximately five weeks.
蛋白异常聚集是阿尔茨海默病(AD)和额颞叶痴呆(FTD)等许多神经退行性疾病的病理细胞标志,其中 tau 蛋白正在聚集,形成神经纤维缠结(NFT),并在神经元之间传播。这些过程与疾病进展和认知功能下降有关。各种治疗方法都旨在预防或减少神经元中的tau聚集。人类诱导多能干细胞(hiPSCs)是神经科学发现中非常有价值的工具,因为它们为体外研究提供了可能无限量的受疾病影响的细胞类型,包括皮质神经元。我们利用 hiPSC 衍生的神经元,经过慢病毒转导后表达易发生聚集的荧光标记版人类 tau 蛋白,生成了 tau 聚集的体外模型。在加入重组声化成对螺旋丝(sPHFs)形式的 tau 种子后,神经元显示出强大的、类似疾病的 tau 蛋白聚集。该模型被开发为一种基于平板的高含量筛选测定法,并与图像分析算法相结合,以评估小分子或基因扰动对 tau 的影响。我们的研究表明,该试验可用于评估小分子或筛选靶向化合物库。通过使用基于 siRNA 的基因敲除,可以评估感兴趣的基因,我们还可以通过在该测定中筛选近 100 种去泛素化酶 (DUB),证明可以筛选靶向基因库。该测定使用基于成像的读数,时间相对较短,可量化 tau 的聚集程度,还能评估细胞活力。此外,它还能很容易地适用于不同的 hiPSC 株系或神经元亚型。总之,这种复杂而高度相关的方法可以每周在多个项目的筛选漏斗中常规应用,产生数据的周转时间约为五周。
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