Inclusive Pattern Generation Protocols to Decode Thiol-Mediated Uptake

Thiol-mediated uptake (TMU) is an intriguing enigma in current chemistry and biology. While the appearance of cell-penetrating activity upon attachment of cascade exchangers (CAXs) has been observed by many and is increasingly being used in practice, the molecular basis of TMU is essentially unknown. The objective of this study was to develop a general protocol to decode the dynamic covalent networks that presumably account for TMU. Uptake inhibition patterns obtained from the removal of exchange partners by either protein knockdown or alternative inhibitors are aligned with original patterns generated by CAX transporters and inhibitors and patterns from alternative functions (here cell motility). These inclusive TMU patterns reveal that the four most significant CAXs known today enter cells along three almost orthogonal pathways. Epidithiodiketopiperazines (ETP) exchange preferably with integrins and protein disulfide isomerases (PDIs), benzopolysulfanes (BPS) with different PDIs, presumably PDIA3, and asparagusic acid (AspA), and antisense oligonucleotide phosphorothioates (OPS) exchange with the transferrin receptor and can be activated by the removal of PDIs with their respective inhibitors. These findings provide a solid basis to understand and use TMU to enable and prevent entry into cells.