Delivery of therapeutic substrates into cells is a fundamental strategy to tackle severe health problems. Unfortunately, cellular membrane strongly shields the cytosol from foreign material entering, posing challenges that must be addressed to develop efficient cellular uptake. Most delivery strategies are based on endocytosis, but most fail because they get trapped and degraded. Thiol-Mediated Uptake (TMU) is a recent method that can deliver different substrates into cells by using thiol-reactive groups that can undergo exchange with thiols and disulfides in the cellular membrane. The continuous exchange allows direct translocation of the substrate of interest into the cytosol, circumventing the necessity of endosomal escape methodology. Although the chemistries have expanded in the last few years not much is known about the undergoing penetration mechanism. In this thesis, we focused on expanding the knowledge on the penetration mechanism, with emphasis on discovering extracellular membrane protein partners.
The first project was inspired by the rich disulfide track existing in integrins which is redox activated and deactivated. Since these receptors control cellular motility, CAXs (cascade exchangers) were used to inhibit this function. Inhibition was in fact confirmed for all the tested CAXs in wound healing and single-cell assays, with epidithiodiketopiperazine S1, dithiabismepanes S2 and Michael acceptors S3 showing enhanced performance compared to the TMU control, DTNB S4, and commercially available integrin and PDI inhibitors (Figure S1). This was the first major step into discovering new proteins partners and provided drug discovery potential for CAXs in integrin-related diseases.
As a result of the success of the first project, we decided to expand the targeting project and develop a new assay to confirm with more certainty the importance of not only integrins but also other receptors and enzymes like protein disulfide isomerase (PDI), epidermal growth factor receptor (EGFR) and scavenger receptor class B type 1 (SCARB1). The ascertain CAX/receptor partnerships, a general protocol based on inactivation was created, either by protein knockdown or chemical inhibition. The patterns generated by these protocols allowed us to expand the asparagusic acid S5 pathway (AspA, transferrin receptor, additionally EGFR and SCARB1-based) but also identify two additional pathways: ETP S1 integrin-based) and benzopolysulfanes S6 (BPS, PDIA3-based), which are nearly orthogonal (Figure S2). By comparing the different patterns generated from the transporters, it was possible to associate them with any of these three pathways, e.g. oligophosphorothioates (OPS) follow closely the AspA S5 pathway since their patterns overlap.
