Supramolecular Entrapment of PTMs and Modulation of Epigenetic Control

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With this project, we would like to evaluate the potential of designed supramolecular binders (clips and tweezers) to interact and interfere with posttranslational modifications (PTMs) of arginine and lysine residues. Basic principles of host-guest complex formation shall be exploited by studying two different functional aspects of clip/tweezer-ligand interaction: entrapment of disease PTMs and reversible modulation of epigenetic control. We shall quantitatively characterize interaction, competition and complex structure formation of supramolecular binders and modifiable residues using a variety of kinetic, biophysical and spectroscopic techniques (NMR, BLI, ITC, Fluorescence-Anisotropy, Absorption Spectroscopy, Biological Kinetic Assays).Within our first objective (PTMs), molecular clips shall be synthesized that enable specific entrapment of different methylated arginine species (MAs) present in human blood. MAs are highly toxic amino acid derivatives that are produced by post-translational modification of proteins and their subsequent proteolysis. Blood level MAs compete with arginine, the substrate of endothelial nitric-oxide synthase (NOS), which is an important activator of muscle relaxation and vessel dilatation. Binding of MAs to NOS impairs its catalytic activity, stiffens the vessel walls and intensifies high blood pressure in human patients. We will modify our clips by click chemistry and attach additional anchors/recognition motifs for the specific arginine N-alkylation pattern. By providing tailored cage-like environments for mono- or dimethylated MA species, we intend to prevent pathologic NOS targeting and restore enzyme activity. Moreover, inclusion of the toxic products inside the clip cavity should accelerate their release from NOS, and thereby enhance renal excretion in vivo. These two effects brought about by clip encapsulation, shall enable serum MA detoxification. In the second objective (Epigenetics), we shall use molecular tweezers to interfere with PTMs of lysine residues in histones. The reversible acetylation of lysines disables attractive histone:DNA interactions and switches transcriptional on and off states in gene expression. Targeting of unmodified lysines on histones by molecular tweezers will reduce their positive charge, thereby imitating the effect of acetylation. This will induce a reversible epigenetic escape from the silenced state by DNA release. To this end, we will create a histone-peptide:DNA-oligomer complex as a model system of a silenced gene, that can be analyzed and monitored in vitro. In parallel, optimized oligomeric tweezers are being developed, which target a well-defined area on the histone peptide. These tweezers are evaluated for their potential to interfere with formation of the peptide:DNA complex and on histone acetyl transferase (HAT) activity.