DARPins bind their cytosolic targets after having been translocated through the protective antigen pore of anthrax toxin.

Intracellular protein-protein interactions in aberrant signaling pathways have emerged as a prime target in several diseases, particularly cancer. Since many protein-protein interactions are mediated by rather flat surfaces, they can typically not be interrupted by small molecules as they require cavities for binding. Therefore, protein drugs might be developed to compete with undesired interactions. However, proteins in general are not able to translocate from the extracellular side to the cytosolic target site by themselves, and thus an efficient protein translocation system, ideally combining efficient translocation with receptor specificity, is in high demand. Anthrax toxin, the tripartite holotoxin of Bacillus anthracis, is one of the best studied bacterial protein toxins and has proven to be a suitable candidate for cell-specific translocation of cargoes in vitro and in vivo. Our group recently developed a retargeted protective antigen (PA) variant fused to different Designed Ankyrin Repeat Proteins (DARPins) to achieve receptor specificity, and we incorporated a receptor domain to stabilize the prepore and prevent cell lysis. This strategy had been shown to deliver high amounts of cargo DARPins fused behind the N-terminal 254 amino acids of Lethal Factor (LFN). Here, we established a cytosolic binding assay, demonstrating the ability of DARPins to refold in the cytosol and bind their target after been translocated by PA.