4.8 Article

Selective cellular probes for mammalian thioredoxin reductase TrxR1: Rational design of RX1, a modular 1,2-thiaselenane redox probe

Journal

CHEM
Volume 8, Issue 5, Pages 1493-1517

Publisher

CELL PRESS
DOI: 10.1016/j.chempr.2022.03.010

Keywords

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Funding

  1. German Research Foundation (DFG) [SFB 1032, SPP 1926, 201269156, SFB TRR 152, 239283807, 426018126, 400324123]
  2. LMUExcellent (Junior Researcher Fund)
  3. Munich Centre for NanoScience initiative (CeNS)
  4. Karolinska Institutet
  5. Knut and Alice Wallenberg Foundations [KAW 2015.0063, KAW 2019.0059]
  6. Swedish Cancer Society [CAN 2018/333, 19 0330 Pj]
  7. Swedish Research Council [2017-01872]
  8. Hungarian Thematic Excellence Programme [TKP2020-NKA-26]
  9. Hungarian National Research, Development and Innovation Office [ED_18-1-2019-0025]
  10. Hungarian National Laboratories Excellence program (under the National Tumor Biology Laboratory project) [NLP-17]
  11. Hungarian Ministry of Human Capacities [AEEK/41872-16/2020]
  12. Intramural Research Program of the National Institutes of Health (NIH), National Center for Advancing Translational Sciences (NCATS)
  13. Fonds der Chemischen Industrie
  14. Studienstiftung des deutschen Volkes
  15. Joachim Herz Foundation
  16. [GRK 2338]
  17. Swedish Research Council [2017-01872] Funding Source: Swedish Research Council

Ask authors/readers for more resources

This study developed a chemical probe for mammalian thioredoxin reductase (TrxR), which is resistant to thiol reductants in cells and releases specific cargos. The probe showed specific sensitivity to cytosolic TrxR1 and performed well in TrxR1-dependent cellular assays. This lays the foundation for in vivo imaging of TrxR1 activity in health and disease, and provides a theoretical basis for the rational design of probes for other key players in redox biology.
Dynamically driven cellular redox networks power a broad range of physiological cellular processes, and additionally are often dysregulated in various pathologies including cancer and inflammatory diseases. Therefore it is vital to be able to image and to respond to the turnover of the key players in redox homeostasis, to understand their physiological dynamics and to target pathological conditions. However, selective modular probes for assessing specific redox enzyme activities in cells are lacking. Here we report the development of cargo-releasing chemical probes that target the mammalian selenoprotein thioredoxin reductase (TrxR) while being fully resistant to thiol reductants in cells, such as the monothiol glutathione (GSH). We used a rationally oriented cyclic selenenylsulfide as a thermodynamically stable and kinetically reversible trigger that matches the chemistry of the unique TrxR active site, and integrated this reducible trigger into modular probes that release arbitrary cargos upon reduction. The probes' redox biochemistry was evaluated over a panel of thiol-type oxidoreductases, particularly showing remarkable, selenocysteine-dependent sensitivity of the RX1 probe design to cytosolic TrxR1, with little response to mitochondrial TrxR2. The probe was cross-validated in cells by TrxR1 knockout, selenium starvation, TrxR1 knock-in, and use of TrxR-selective chemical inhibitors, showing excellent TrxR1-dependent cellular performance. The RX1 design is therefore a robust, cellularly-validated, modular probe system for mammalian TrxR1. This sets the stage for in vivo imaging of TrxR1 activity in health and disease; and the thermodynamic and kinetic considerations behind its selectivity mechanism represent a significant advance towards rationally-designed probes for other key players in redox biology.

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