Regulation of eDHFR-tagged proteins with trimethoprim PROTACs
Abstract Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-drive...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2023-11-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-023-42820-3 |
| _version_ | 1797636806378782720 |
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| author | Jean M. Etersque Iris K. Lee Nitika Sharma Kexiang Xu Andrew Ruff Justin D. Northrup Swarbhanu Sarkar Tommy Nguyen Richard Lauman George M. Burslem Mark A. Sellmyer |
| author_facet | Jean M. Etersque Iris K. Lee Nitika Sharma Kexiang Xu Andrew Ruff Justin D. Northrup Swarbhanu Sarkar Tommy Nguyen Richard Lauman George M. Burslem Mark A. Sellmyer |
| author_sort | Jean M. Etersque |
| collection | DOAJ |
| description | Abstract Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo. |
| first_indexed | 2024-03-11T12:39:27Z |
| format | Article |
| id | doaj.art-37f8ccd04b6741fe96a85487d62b0e1e |
| institution | Directory Open Access Journal |
| issn | 2041-1723 |
| language | English |
| last_indexed | 2024-03-11T12:39:27Z |
| publishDate | 2023-11-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj.art-37f8ccd04b6741fe96a85487d62b0e1e2023-11-05T12:22:41ZengNature PortfolioNature Communications2041-17232023-11-0114111510.1038/s41467-023-42820-3Regulation of eDHFR-tagged proteins with trimethoprim PROTACsJean M. Etersque0Iris K. Lee1Nitika Sharma2Kexiang Xu3Andrew Ruff4Justin D. Northrup5Swarbhanu Sarkar6Tommy Nguyen7Richard Lauman8George M. Burslem9Mark A. Sellmyer10Department of Radiology, Perelman School of Medicine, University of PennsylvaniaDepartment of Radiology, Perelman School of Medicine, University of PennsylvaniaDepartment of Radiology, Perelman School of Medicine, University of PennsylvaniaDepartment of Radiology, Perelman School of Medicine, University of PennsylvaniaDepartment of Radiology, Perelman School of Medicine, University of PennsylvaniaDepartment of Radiology, Perelman School of Medicine, University of PennsylvaniaDepartment of Radiology, Perelman School of Medicine, University of PennsylvaniaDepartment of Radiology, Perelman School of Medicine, University of PennsylvaniaThe Department of Biochemistry and Biophysics, Perelman School of Medicine, University of PennsylvaniaThe Department of Biochemistry and Biophysics, Perelman School of Medicine, University of PennsylvaniaDepartment of Radiology, Perelman School of Medicine, University of PennsylvaniaAbstract Temporal control of protein levels in cells and living animals can be used to improve our understanding of protein function. In addition, control of engineered proteins could be used in therapeutic applications. PRoteolysis-TArgeting Chimeras (PROTACs) have emerged as a small-molecule-driven strategy to achieve rapid, post-translational regulation of protein abundance via recruitment of an E3 ligase to the target protein of interest. Here, we develop several PROTAC molecules by covalently linking the antibiotic trimethoprim (TMP) to pomalidomide, a ligand for the E3 ligase, Cereblon. These molecules induce degradation of proteins of interest (POIs) genetically fused to a small protein domain, E. coli dihydrofolate reductase (eDHFR), the molecular target of TMP. We show that various eDHFR-tagged proteins can be robustly degraded to 95% of maximum expression with PROTAC molecule 7c. Moreover, TMP-based PROTACs minimally affect the expression of immunomodulatory imide drug (IMiD)-sensitive neosubstrates using proteomic and biochemical assays. Finally, we show multiplexed regulation with another known degron-PROTAC pair, as well as reversible protein regulation in a rodent model of metastatic cancer, demonstrating the formidable strength of this system. Altogether, TMP PROTACs are a robust approach for selective and reversible degradation of eDHFR-tagged proteins in vitro and in vivo.https://doi.org/10.1038/s41467-023-42820-3 |
| spellingShingle | Jean M. Etersque Iris K. Lee Nitika Sharma Kexiang Xu Andrew Ruff Justin D. Northrup Swarbhanu Sarkar Tommy Nguyen Richard Lauman George M. Burslem Mark A. Sellmyer Regulation of eDHFR-tagged proteins with trimethoprim PROTACs Nature Communications |
| title | Regulation of eDHFR-tagged proteins with trimethoprim PROTACs |
| title_full | Regulation of eDHFR-tagged proteins with trimethoprim PROTACs |
| title_fullStr | Regulation of eDHFR-tagged proteins with trimethoprim PROTACs |
| title_full_unstemmed | Regulation of eDHFR-tagged proteins with trimethoprim PROTACs |
| title_short | Regulation of eDHFR-tagged proteins with trimethoprim PROTACs |
| title_sort | regulation of edhfr tagged proteins with trimethoprim protacs |
| url | https://doi.org/10.1038/s41467-023-42820-3 |
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