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Regulation of eDHFR-tagged proteins with trimethoprim PROTACs

Author

Listed:
  • Jean M. Etersque

    (University of Pennsylvania)

  • Iris K. Lee

    (University of Pennsylvania)

  • Nitika Sharma

    (University of Pennsylvania)

  • Kexiang Xu

    (University of Pennsylvania)

  • Andrew Ruff

    (University of Pennsylvania)

  • Justin D. Northrup

    (University of Pennsylvania)

  • Swarbhanu Sarkar

    (University of Pennsylvania)

  • Tommy Nguyen

    (University of Pennsylvania)

  • Richard Lauman

    (University of Pennsylvania)

  • George M. Burslem

    (University of Pennsylvania
    University of Pennsylvania)

  • Mark A. Sellmyer

    (University of Pennsylvania
    University of Pennsylvania)

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.

Suggested Citation

  • 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, 2023. "Regulation of eDHFR-tagged proteins with trimethoprim PROTACs," Nature Communications, Nature, vol. 14(1), pages 1-15, December.
  • Handle: RePEc:nat:natcom:v:14:y:2023:i:1:d:10.1038_s41467-023-42820-3
    DOI: 10.1038/s41467-023-42820-3
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