Peptides hormones play an important role in the regulation of manifold activities in the body. Many of them transmit their activity through G-Protein Coupled Receptors (GPCR), which are among the most promising drug targets nowadays. Accordingly, elucidating the binding mode of ligands is essential. Whereas several small molecule systems have been well characterized, ligand binding of large and flexible peptides is still more challenging. In addition to ligand binding and receptor activation, indirect mechanisms have been shown to play a role for drugs addressing GPCRs. This includes desensitization, internalization and accordingly their potential use as drug shuttles, e.g. in tumor targeting. Accordingly, in addition to ligand binding, internalization has to be addressed and to be studied, including arrestin recruitment. Accordingly, ligand binding, structural dynamics, and internalization have to be addressed and to be studied to address G protein-coupled receptors for drug development. The neuropeptide Y/pancreatic polypeptide family contains 36 amino acid peptides that bind in human to four different so-called Y-receptors. By a combination of X-ray analysis, NMR, molecular modeling and cross-linking combined with mass spectrometry, we could recently identify the distinct binding modes of NPY to the Y1- and the Y2 receptors. We could further demonstrate that chemical modification of the ligand, including fluorescence labeling, lipidization, and PEGylation significantly modifies the trafficking of the ligand. By labeling of the receptor with a novel template-assisted ligation strategy, we can follow ligand/receptor complexes in living cells. Furthermore, we identified a different mode of arrestin binding and recruitment. Neuropeptide Y1 and Y2 receptors have been shown to play a relevant role in different tumors. In breast cancer we demonstrated that human Y1 receptors are addressable by peptide conjugates using 99mTc or 18F PET-tracers. We now designed Y1 receptor selective peptides linked to different toxophors. Furthermore, we characterized the mechanism of direct and peptide-mediated uptake of tubulysin-related toxins. In the field of tumor therapy, peptide-drug conjugates are already well accepted. However, the concept of receptormediated internalization and subsequent tissue specific intracellular application is not limited to the selective addressing of tumors. This may open up a new field of targeted therapy by mid-sized drugs.
