NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation

Nat Methods. 2022 Feb;19(2):205-215. doi: 10.1038/s41592-021-01364-4. Epub 2022 Feb 7.

Abstract

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Genetically Modified
  • CHO Cells
  • Cricetulus
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Metronidazole / pharmacokinetics
  • Metronidazole / pharmacology*
  • Nitroreductases / chemistry
  • Nitroreductases / genetics
  • Nitroreductases / metabolism*
  • Prodrugs / chemistry*
  • Prodrugs / pharmacology
  • Protein Engineering / methods
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Retina / cytology
  • Retina / drug effects
  • Vibrio / enzymology
  • Zebrafish / genetics

Substances

  • Prodrugs
  • Recombinant Proteins
  • Metronidazole
  • Green Fluorescent Proteins
  • Nitroreductases