Inhibition of Thiol‐Mediated Uptake with Irreversible Covalent Inhibitors

Abstract

Thiol‐mediated uptake is emerging as method of choice to penetrate cells. This study focuses on irreversible covalent inhibitors of thiol‐mediated uptake. High‐content high‐throughput screening of the so far largest collection of hypervalent iodine reagents affords inhibitors that are more than 250 times more active than Ellman’s reagent and rival the best dynamic covalent inhibitors. Comparison with other irreversible reagents reveals that inhibition within one series follows reactivity, whereas inhibition across series deviates from reactivity. These trends support that molecular recognition, besides dynamic covalent exchange, contributes significantly to thiol‐mediated uptake. The most powerful inhibitors besides the best hypervalent iodine reagents were Fukuyama’s nosyl protecting group and super‐cinnamaldehydes that have been introduced as irreversible activators of the pain receptor TRPA1. Considering that several viruses use different forms of thiol‐mediated uptake to enter cells, the identification of new irreversible inhibitors of thiol‐mediated uptake is of general interest for the discovery of new antivirals.

Cyclic Thiosulfonates for Thiol-Mediated Uptake: Cascade Exchangers, Transporters, Inhibitors

“…OPS 32 was best inhibited by the irreversible hypervalent iodine reagent 49 (entry 35, Figure 8 ). 52 , 79 Like CTOs “self-inhibiting” CTO 28 , the cytosolic delivery of ETP 31 was best self-inhibited with ETPs, with the expanded tetrasulfide 47 being most interesting (entry 30). The resulting heatmap showed strong contrasts, powerful self-inhibition, and different top inhibitors for all transporters ( Figure 8 ).…”

“…This high concentration effect was as previously observed in the screening of thiol-mediated uptake inhibitors, also with the popular Ellman reagent 16 . 51 , 79 …”

“…The more hydrophilic, anionic ebselen analogue 45 was more active and nontoxic at 50 μM (entry 6). The hypervalent iodine reagent 49 is an irreversible covalent thiol-reactive agent, and was the most active inhibitor of cytosolic delivery of OPS 32 but less powerful against ETP 31 ( 79 ) and particularly CTO 28 ( Figure 8 ). The covalent reagent 49 did not inhibit SC2 entry at 5 μM, i.e., around the IC 50 of dimers 4 and 33 (entry 17).…”

“…This high concentration effect was as previously observed in the screening of thiol-mediated uptake inhibitors, also with the popular Ellman reagent 16. 51,79 The de-silylated dimer 41 was inactive at 5 μM (entry 15), i.e., around the IC 50 of dimer 4 (entry 1, Figure 9A). This result echoed the poorer activity of transporter 29 compared to 28 (Figure 7).…”

Cyclic 5-membered disulfides are not selective substrates of thioredoxin reductase, but are opened nonspecifically

“…2a ) 26 , 29 – 35 . It will be important to note that this process may be strongly affected by treatment with thiol-reactive species: cellular uptake rates are decreased many-fold by thiol-reactive electrophiles or oxidants; or instead enhanced by reducing agents 30 , 36 , 37 . Studying the role of membrane thiols in this process, and their connection to the intracellular redox environment, is also an expanding topic 38 – 42 .…”