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NTR 2.0: a rationally engineered prodrug-converting enzyme with substantially enhanced efficacy for targeted cell ablation.
Sharrock, Abigail V; Mulligan, Timothy S; Hall, Kelsi R; Williams, Elsie M; White, David T; Zhang, Liyun; Emmerich, Kevin; Matthews, Frazer; Nimmagadda, Saumya; Washington, Selena; Le, Katherine D; Meir-Levi, Danielle; Cox, Olivia L; Saxena, Meera T; Calof, Anne L; Lopez-Burks, Martha E; Lander, Arthur D; Ding, Ding; Ji, Hongkai; Ackerley, David F; Mumm, Jeff S.
Affiliation
  • Sharrock AV; School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
  • Mulligan TS; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Hall KR; School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
  • Williams EM; School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand.
  • White DT; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Zhang L; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Emmerich K; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Matthews F; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Nimmagadda S; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Washington S; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Le KD; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Meir-Levi D; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Cox OL; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Saxena MT; Department of Ophthalmology, Wilmer Eye Institute, Johns Hopkins University, Baltimore, MD, USA.
  • Calof AL; Luminomics, Baltimore, MD, USA.
  • Lopez-Burks ME; Department of Anatomy and Neurobiology, University of California, Irvine, CA, USA.
  • Lander AD; Department of Developmental and Cell Biology, University of California, Irvine, CA, USA.
  • Ding D; Center for Complex Biological Systems, University of California, Irvine, CA, USA.
  • Ji H; Department of Developmental and Cell Biology, University of California, Irvine, CA, USA.
  • Ackerley DF; Center for Complex Biological Systems, University of California, Irvine, CA, USA.
  • Mumm JS; Department of Developmental and Cell Biology, University of California, Irvine, CA, USA.
Nat Methods ; 19(2): 205-215, 2022 02.
Article in En | MEDLINE | ID: mdl-35132245
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.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Nitroreductases / Prodrugs / Metronidazole Limits: Animals / Humans Language: En Journal: Nat Methods Journal subject: TECNICAS E PROCEDIMENTOS DE LABORATORIO Year: 2022 Type: Article Affiliation country: New Zealand
Fulltext
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Nitroreductases / Prodrugs / Metronidazole Limits: Animals / Humans Language: En Journal: Nat Methods Journal subject: TECNICAS E PROCEDIMENTOS DE LABORATORIO Year: 2022 Type: Article Affiliation country: New Zealand