The "sneaking-ligand" approach: cell-type specific inhibition of the classical NF-κB pathway.

The intracellular delivery of molecules across the plasma membrane represents a major obstacle. The conjugation of cell-permeable peptides (CPPs) to proteins promotes the uptake and internalization. However, uptake of CPPs is receptor independent and not cell-type specific. Recently, we established the "sneaking-ligand" approach which is based on multimodular recombinant fusion proteins that consist of three modules connected with serine-glycine linkers. Module one is responsible for receptor-mediated endocytosis; module two supports translocation into the cytoplasm so that the effector module three can interact with its binding partner in the cytoplasm. For NF-κB inhibition, we described an NF-κB inhibitor that targets selectively the activated endothelium via an oligopeptide motif. Upon E-selectin-mediated endocytosis, the Pseudomonas exotoxin A domain II (ETAII) translocates the NEMO-binding peptide to the cytoplasm interfering with IκB kinase complex assembly. Inflammatory autoimmune diseases are triggered, but also resolved by a variety of cell types. Therefore, the inhibition of NF-κB should be restricted to those cells that are crucially involved in the pathogenesis of inflammatory diseases. A general blockade of NF-κB may result in severe immunosuppression and possibly in organ dysfunction or damage. The "sneaking-ligand" approach could minimize the risks of therapeutic interventions and identify disease-relevant cell types. Here we describe the recombinant expression and purification of the E-selectin-specific "sneaking-ligand construct" (SLC1) and its ability to inhibit cytokine-induced NF-κB activation in vitro.