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

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.

Cyclic dichalcogenides extend the reach of bioreductive prodrugs to harness the thioredoxin system: applications toseco-duocarmycins

“…This design also separates the functional steps responsible for bioactivity ( Fig 2c ): (1) bioreduction of the trigger by dithiol or selenolthiol reductases e.g. Trx 17 or TrxR 18 ; (2) 5-exo-trig cyclisation by the trigger to release the (biologically inactive) duocarmycin phenolic cargo; (3) 1,4-nucleophilic Winstein cyclisation 35 creates the activated cyclopropane CBI*; (4) CBI* docks in the DNA minor groove while twisting its aryl-aryl plane, and the now more activated cyclopropane irreversibly alkylates adenosine at the N3-position; 36 (5) the quaternised purine base is spontaneously excised, causing DNA damage and ultimately cell death.…”

“…1 st generation Trx-triggers SS60 and solubilised P-SS60 ; 17 2 nd generation solubilised bicyclic Trx-triggers Me-SS66T, Me-SS66C and P-SS66C ; 19 TrxR-trigger SeS60 18 . “Upper limit” control triggers: very labile MC , monothiol-labile SS00 M .…”

“…NH 4 HCO 2 as the hydrogen source, as reported by Major 28 , to avoid the unwanted naphthalene hydrogenation and dechlorination seen with H 2 (g) . The phenolic chloroformate produced by reaction with in situ generated phosgene was carbamylated 17,18 with the eight trigger secondary amines giving good to excellent yields of trigger-CBI intermediates (e.g. trigger H-SS66C-H ( 2 ) giving intermediate H-SS66C-CBI-Boc ( 3 ), Fig 4 ).…”

“…Trx, and/or TrxR, Grx, etc.). 17,18 These performance features had not been tested with naphthols, so we aimed to use HPLC to confirm them while also testing their reduction-based activation sequence ( Fig 2c and Fig S4-S6 ).…”

“…19 Cyclic selenenylsulfides instead had a selenium-preferring, regioisomer-dependent activation mechanism making them excellently selective substrates for TrxR1 in live cells. 18 These bioreductive motifs were validated in fluorogenic probes in acute applications (minutes to hours) in cell culture. However, it is unknown whether such designs can be useful for long-term redox-selective drug delivery, particularly in the context of cancer for which their Trx/TrxR targets’ biology is most relevant ( Fig 1b ), since in vivo uses are more stringent: e.g.…”