Amyloidoses are diseases characterized by abnormal protein folding and self-assembly, for which no cure is available. Inhibition or modulation of abnormal protein self-assembly therefore is an attractive strategy for prevention and treatment of amyloidoses. We examined Lys-specific molecular tweezers and discovered a lead compound termed CLR01, which is capable of inhibiting the aggregation and toxicity of multiple amyloidogenic proteins by binding to Lys residues and disrupting hydrophobic and electrostatic interactions important for nucleation, oligomerization, and fibril elongation. Importantly, CLR01 shows no toxicity at concentrations substantially higher than those needed for inhibition. We used amyloid β-protein (Aβ) to further explore the binding site(s) of CLR01 and the impact of its binding on the assembly process. Mass-spectrometry and solution-state NMR demonstrated binding of CLR01 to the Lys residues in Aβ at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aβ oligomers but were non-toxic and were not recognized by the oligomer-specific antibody A11. Thus, CLR01 binds already at the monomer stage and modulates the assembly reaction into formation of non-toxic structures. The data suggest that molecular tweezers are unique, process-specific inhibitors of aberrant protein aggregation and toxicity, which hold promise for developing disease-modifying therapy for amyloidoses.

Supramolecular chemistry has recently emerged as a promising way to modulate protein functions, but devising molecules that will interact with a protein in the desired manner is difficult as many competing interactions exist in a biological environment (with solvents, salts or different sites for the target biomolecule). We now show that lysine-specific molecular tweezers bind to a 14-3-3 adapter protein and modulate its interaction with partner proteins. The tweezers inhibit binding between the 14-3-3 protein and two partner proteins--a phosphorylated (C-Raf) protein and an unphosphorylated one (ExoS)--in a concentration-dependent manner. Protein crystallography shows that this effect arises from the binding of the tweezers to a single surface-exposed lysine (Lys214) of the 14-3-3 protein in the proximity of its central channel, which normally binds the partner proteins. A combination of structural analysis and computer simulations provides rules for the tweezers' binding preferences, thus allowing us to predict their influence on this type of protein-protein interactions.

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The molecular tweezers (1, 2) and clips (3-7) containing naphthalene and benzene spacer units can be synthesized via repetitive Diels-Alder reactions by the use of a molecular "Lego" set consisting of bisdienophiles (8, 9, 14) and dienes (10, 13). The new receptors selectively bind electron-deficient neutral and cationic substrates in solution. Only the benzene-spaced tweezers form complexes with aliphatic substrates, whereas the other receptors bind aromatic substrates preferentially. HPLC studies with 1 and 2 chemically bonded to stationary phases give similar results for the heterogeneous systems. The formation of stable complexes between the water-soluble clip 5g and N-alkylpyridinium cations, such as N-methylnicotinamide and NAD(+), in aqueous solution illustrates the importance of the hydrophobic effect for arene-arene interactions. The dynamics of the complex formation and substrate mobility were investigated by the use of temperature-dependent liquid- and solid-state NMR spectroscopy. The electrostatic potential surface (EPS) of 1-7 is calculated to be surprisingly negative on the concave side of each molecule and, hence, complementary to the EPS of the electron-deficient substrates, suggesting that the attractive receptor-substrate interaction is here of predominantly electrostatic nature.

Lysine and arginine play a key role in numerous biological recognition processes controlling, inter alia, gene regulation, glycoprotein targeting and vesicle transport. They are also found in signaling peptide sequences responsible, e.g. for bacterial cell wall biosynthesis, Alzheimer peptide aggregation and skin regeneration. Almost none of all artificial receptor structures reported to date are selective and efficient for lysine residues in peptides or proteins. An artificial molecular tweezer is introduced which displays an exceptionally high affinity for lysine (K(a) approximately 5000 in neutral phosphate buffer). It features an electron-rich torus-shaped cavity adorned with two peripheral anionic phosphonate groups. Exquisite selectivity for arginine and lysine is achieved by threading the whole amino acid side chain through the cavity and subsequent locking by formation of a phosphonate-ammonium/guanidinium salt bridge. This pseudorotaxane-like geometry is also formed in small basic signaling peptides, which can be bound with unprecedented affinity in buffered aqueous solution. NMR titrations, NOESY and VT experiments as well as ITC measurements and Monte Carlo simulations unanimously point to an enthalpy-driven process utilizing a combination of van der Waals interactions and substantial electrostatic contributions for a conformational lock. Since DMSO and acetonitrile compete with the amino acid guest inside the cavity, a simple change in the cosolvent composition renders the whole complexation process reversible.