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

09 July 2021, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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.

Keywords

Thioredoxin reductase
Chemical probes
Selenenyl sulfides
Redox probes
Reduction triggers
Thioredoxin
Fluorogenic probes
Probe design
Selenium
Redox biology
disulfide
chemical biology
molecular probe
imaging
cell biology
biochemistry
redox biochemistry
reductive probe
bioreduction
enzyme selectivity
artificial substrate
diselenide
glutaredoxin
glutathione
glutathione reductase
reductase
nadph
dithiol
selenolthiol
selenenylsulfide
prodrug

Supplementary materials

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Supporting Information
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Materials and Methods, Synthesis, Supplementary Notes, Analytical data
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