A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms
The regulation of cell physiology depends largely upon interactions of functionally distinct proteins and cellular components. These interactions may be transient or long-lived, but often affect protein motion. Measurement of protein dynamics within a cellular environment, particularly while perturb...
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| Language: | English |
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eLife Sciences Publications Ltd
2025-01-01
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| Online Access: | https://elifesciences.org/articles/93183 |
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| author | David Trombley McSwiggen Helen Liu Ruensern Tan Sebastia Agramunt Puig Lakshmi B Akella Russell Berman Mason Bretan Hanzhe Chen Xavier Darzacq Kelsey Ford Ruth Godbey Eric Gonzalez Adi Hanuka Alec Heckert Jaclyn J Ho Stephanie L Johnson Reed Kelso Aaron Klammer Ruchira Krishnamurthy Jifu Li Kevin Lin Brian Margolin Patrick McNamara Laurence Meyer Sarah E Pierce Akshay Sule Connor Stashko Yangzhong Tang Daniel J Anderson Hilary P Beck |
| author_facet | David Trombley McSwiggen Helen Liu Ruensern Tan Sebastia Agramunt Puig Lakshmi B Akella Russell Berman Mason Bretan Hanzhe Chen Xavier Darzacq Kelsey Ford Ruth Godbey Eric Gonzalez Adi Hanuka Alec Heckert Jaclyn J Ho Stephanie L Johnson Reed Kelso Aaron Klammer Ruchira Krishnamurthy Jifu Li Kevin Lin Brian Margolin Patrick McNamara Laurence Meyer Sarah E Pierce Akshay Sule Connor Stashko Yangzhong Tang Daniel J Anderson Hilary P Beck |
| author_sort | David Trombley McSwiggen |
| collection | DOAJ |
| description | The regulation of cell physiology depends largely upon interactions of functionally distinct proteins and cellular components. These interactions may be transient or long-lived, but often affect protein motion. Measurement of protein dynamics within a cellular environment, particularly while perturbing protein function with small molecules, may enable dissection of key interactions and facilitate drug discovery; however, current approaches are limited by throughput with respect to data acquisition and analysis. As a result, studies using super-resolution imaging are typically drawing conclusions from tens of cells and a few experimental conditions tested. We addressed these limitations by developing a high-throughput single-molecule tracking (htSMT) platform for pharmacologic dissection of protein dynamics in living cells at an unprecedented scale (capable of imaging >106 cells/day and screening >104 compounds). We applied htSMT to measure the cellular dynamics of fluorescently tagged estrogen receptor (ER) and screened a diverse library to identify small molecules that perturbed ER function in real time. With this one experimental modality, we determined the potency, pathway selectivity, target engagement, and mechanism of action for identified hits. Kinetic htSMT experiments were capable of distinguishing between on-target and on-pathway modulators of ER signaling. Integrated pathway analysis recapitulated the network of known ER interaction partners and suggested potentially novel, kinase-mediated regulatory mechanisms. The sensitivity of htSMT revealed a new correlation between ER dynamics and the ability of ER antagonists to suppress cancer cell growth. Therefore, measuring protein motion at scale is a powerful method to investigate dynamic interactions among proteins and may facilitate the identification and characterization of novel therapeutics. |
| format | Article |
| id | doaj-art-af3c50cd17f8439887f023cdd95f439f |
| institution | Kabale University |
| issn | 2050-084X |
| language | English |
| publishDate | 2025-01-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj-art-af3c50cd17f8439887f023cdd95f439f2025-01-09T15:49:41ZengeLife Sciences Publications LtdeLife2050-084X2025-01-011210.7554/eLife.93183A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanismsDavid Trombley McSwiggen0Helen Liu1Ruensern Tan2Sebastia Agramunt Puig3Lakshmi B Akella4Russell Berman5Mason Bretan6Hanzhe Chen7Xavier Darzacq8https://orcid.org/0000-0003-2537-8395Kelsey Ford9Ruth Godbey10Eric Gonzalez11Adi Hanuka12Alec Heckert13Jaclyn J Ho14Stephanie L Johnson15Reed Kelso16Aaron Klammer17Ruchira Krishnamurthy18Jifu Li19Kevin Lin20Brian Margolin21https://orcid.org/0000-0003-3365-7677Patrick McNamara22https://orcid.org/0000-0003-2756-0887Laurence Meyer23Sarah E Pierce24Akshay Sule25Connor Stashko26Yangzhong Tang27Daniel J Anderson28Hilary P Beck29https://orcid.org/0000-0002-5003-1361Eikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United States; University of California, Berkeley, Berkeley, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesEikon Therapeutics Inc, Hayward, United StatesThe regulation of cell physiology depends largely upon interactions of functionally distinct proteins and cellular components. These interactions may be transient or long-lived, but often affect protein motion. Measurement of protein dynamics within a cellular environment, particularly while perturbing protein function with small molecules, may enable dissection of key interactions and facilitate drug discovery; however, current approaches are limited by throughput with respect to data acquisition and analysis. As a result, studies using super-resolution imaging are typically drawing conclusions from tens of cells and a few experimental conditions tested. We addressed these limitations by developing a high-throughput single-molecule tracking (htSMT) platform for pharmacologic dissection of protein dynamics in living cells at an unprecedented scale (capable of imaging >106 cells/day and screening >104 compounds). We applied htSMT to measure the cellular dynamics of fluorescently tagged estrogen receptor (ER) and screened a diverse library to identify small molecules that perturbed ER function in real time. With this one experimental modality, we determined the potency, pathway selectivity, target engagement, and mechanism of action for identified hits. Kinetic htSMT experiments were capable of distinguishing between on-target and on-pathway modulators of ER signaling. Integrated pathway analysis recapitulated the network of known ER interaction partners and suggested potentially novel, kinase-mediated regulatory mechanisms. The sensitivity of htSMT revealed a new correlation between ER dynamics and the ability of ER antagonists to suppress cancer cell growth. Therefore, measuring protein motion at scale is a powerful method to investigate dynamic interactions among proteins and may facilitate the identification and characterization of novel therapeutics.https://elifesciences.org/articles/93183drug discoverysingle-molecule imagingestrogen receptorhigh-throughput imaginglive-cell imagingprotein motion |
| spellingShingle | David Trombley McSwiggen Helen Liu Ruensern Tan Sebastia Agramunt Puig Lakshmi B Akella Russell Berman Mason Bretan Hanzhe Chen Xavier Darzacq Kelsey Ford Ruth Godbey Eric Gonzalez Adi Hanuka Alec Heckert Jaclyn J Ho Stephanie L Johnson Reed Kelso Aaron Klammer Ruchira Krishnamurthy Jifu Li Kevin Lin Brian Margolin Patrick McNamara Laurence Meyer Sarah E Pierce Akshay Sule Connor Stashko Yangzhong Tang Daniel J Anderson Hilary P Beck A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms eLife drug discovery single-molecule imaging estrogen receptor high-throughput imaging live-cell imaging protein motion |
| title | A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms |
| title_full | A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms |
| title_fullStr | A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms |
| title_full_unstemmed | A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms |
| title_short | A high-throughput platform for single-molecule tracking identifies drug interaction and cellular mechanisms |
| title_sort | high throughput platform for single molecule tracking identifies drug interaction and cellular mechanisms |
| topic | drug discovery single-molecule imaging estrogen receptor high-throughput imaging live-cell imaging protein motion |
| url | https://elifesciences.org/articles/93183 |
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