Non-antibiotic Small-Molecule Regulation of DHFR-Based Destabilizing Domains In Vivo

Hui Peng, Viet Q. Chau, Wanida Phetsang, Rebecca M. Sebastian, M. Rhia L. Stone, Shyamtanu Datta, Marian Renwick, Yusuf T. Tamer, Erdal Toprak, Andrew Y. Koh, Mark A.T. Blaskovich, John D. Hulleman

Research output: Contribution to journalArticlepeer-review

13 Scopus citations


The E. coli dihydrofolate reductase (DHFR) destabilizing domain (DD), which shows promise as a biologic tool and potential gene therapy approach, can be utilized to achieve spatial and temporal control of protein abundance in vivo simply by administration of its stabilizing ligand, the routinely prescribed antibiotic trimethoprim (TMP). However, chronic TMP use drives development of antibiotic resistance (increasing likelihood of subsequent infections) and disrupts the gut microbiota (linked to autoimmune and neurodegenerative diseases), tempering translational excitement of this approach in model systems and for treating human diseases. Herein, we identified a TMP-based, non-antibiotic small molecule, termed 14a (MCC8529), and tested its ability to control multiple DHFR-based reporters and signaling proteins. We found that 14a is non-toxic and can effectively stabilize DHFR DDs expressed in mammalian cells. Furthermore, 14a crosses the blood-retinal barrier and stabilizes DHFR DDs expressed in the mouse eye with kinetics comparable to that of TMP (≤6 h). Surprisingly, 14a stabilized a DHFR DD in the liver significantly better than TMP did, while having no effect on the mouse gut microbiota. Our results suggest that alternative small-molecule DHFR DD stabilizers (such as 14a) may be ideal substitutes for TMP in instances when conditional, non-antibiotic control of protein abundance is desired in the eye and beyond.

Original languageEnglish (US)
Pages (from-to)27-39
Number of pages13
JournalMolecular Therapy - Methods and Clinical Development
StatePublished - Dec 13 2019
Externally publishedYes

Bibliographical note

Publisher Copyright:
© 2019 The Author(s)


  • chemical biology
  • conditional regulation
  • destabilizing domain
  • gene therapy
  • hepatic
  • non-antibiotic
  • ocular
  • trimethoprim


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