Toward a Traceless Tag for the Thiol‐Mediated Uptake of Proteins

Abstract

The emergence of thiol‐mediated uptake (TMU) as a powerful strategy to penetrate cells calls for the development of practical small‐molecule TMU tools for traceless delivery. Toward this goal, esters are explored here as bioreversible linkers between dynamic covalent cascade exchangers accounting for TMU and the protein of interest (POI). The method relies on α‐aryl‐α‐diazoamides that react with carboxylic acids of the POI to form esters that can be enzymatically hydrolyzed inside cells to release the native POI. A two‐step protocol is established for bioreversible conjugation of TMU tags to the POI. Despite the small number of tags attached to POIs to prevent isoelectric precipitation, POIs with traceless TMU tags are shown to efficiently enter cells not only in 2D culture but also in 3D spheroids mimicking deep tissue, confirming a key advantage of TMU. Uptake inhibition by various thiol‐reactive agents confirms the participation of cell‐surface thiols in cell penetration, i. e., the occurrence of TMU.

Inclusive Pattern Generation Protocols to Decode Thiol-Mediated Uptake

“…This finding was intriguing because the transferrin receptor has also been implicated in viral entry . Efficient transcytosis, needed to deliver iron to the brain, could explain why TMU of AspA conjugates excels at penetrating deep tissue. ,, …”

“…Recent results with cellpenetrating GFP-AspA conjugates were similar to the patterns generated with Fl-AspA (Figure 4a), with five out of seven matching inhibitors, including the top two. 12 Nevertheless, existing reports on SOI dependence of the absolute activity of CAX suggest that pattern generation will also turn out to be SOI dependent to an extent that remains to be explored CAX by CAX.…”

“…1 Efficient transcytosis, needed to deliver iron to the brain, could explain why TMU of AspA conjugates excels at penetrating deep tissue. 12,20,35 In this study, the knockdown of integrins was selected to uncover their role in TMU, as implied by our recent studies on cell motility (P2, Figure 2b). 38 This was interesting because integrins are not only responsible for cell adhesion and motility but also involved in signal transduction, viral entry, thrombosis, and tumor progression.…”

“…TMU has long been known from eclectic observations but without much follow-up. , In recent years, however, a rapidly growing number of reports have demonstrated their power in advanced applications. Namely, TMU works also in vivo, i.e., animals (up to genome editing), ,,,, plants, and bacteria, delivers well into deep tissue, ,, and accounts for the cell-penetrating ability of antisense oligophosphorothioates (OPS, Figure b). − Moreover, TMU inhibitors also attenuate the cellular entry of pathogens, including several viruses, , and the cell motility . These activities suggest that TMU is relevant not only for drug delivery but also for drug discovery.…”

“…1 It is understood that the TMU can operate in combination with different uptake mechanisms. Examples exist for membrane fusion, 24 including the first observation during viral entry, 36 endocytosis, 12,13,15,18,20,22,35 and mostly direct penetration through the plasma membrane. 1 Accounting also for most endosomal escape, the problematic step during endocytosis, direct penetration, has attracted most attention for the cytosolic delivery of SOIs by TMU 1 (the shift from endosomal capture to cytosolic delivery upon activation of TMU has been exemplified with OPS 22 ).…”