Frank-Gerrit Klärner

Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins

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 nontoxic 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 nontoxic 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.

Aromatic interactions by molecular tweezers and clips in chemical and biological systems.

Noncovalent interactions involving aromatic rings, such as π-stacking and CH-π, occur throughout a range of fundamental processes including self-assembly and (bio)catalysis. Molecular clips and tweezers possess a central parallel or torus-shaped cavity with a surrounding belt of convergent aromatic rings; hence these structures exploit multiple aromatic interactions in a positively cooperative manner. Both clips and tweezers demonstrate selective binding of cationic or neutral guests that bear acceptor groups. The electrostatic surface potentials (ESP) explain this unexpected behavior: calculated ESPs were highly negative inside the tweezer or clip cavity, providing complementary profiles to the positive ESP plots of their preferred guest molecules. This Account presents more complex systems that use aromatic clips and tweezers to alter the reactivities of included guest species, to distinguish between guest enantiomers, and to interfere with biological processes such as enzymatic activity and protein aggregation. Napthalene tweezers show potential applications in organocatalysis. When pyridinium moieties are bound within the spacious cavity of naphthyl-spaced tweezers, the resulting complex significantly influences the first step of single-electron reductions of (bi)pyridinium salts. In addition, the environment within the tweezer cavity strongly accelerates the Menshutkin reaction (the alkylation of pyridine derivatives). Introduction of phosphonate, phosphate, or sulfate anions into the central aromatic bridge renders clips and tweezers water-soluble. Larger systems form extremely tight intertwined dimers that rely on the nonclassical hydrophobic effect for their stability. Smaller clips and tweezers with a simple benzene bridge remain monomeric in buffered aqueous solution and display a complementary binding profile. While the clips with parallel sidewalls prefer flat aromatic cations such as pyridinium salts, the torus-shaped tweezers bind to basic amino acids lysine and arginine via a threading process. These mutually exclusive binding modes make water-soluble clips and tweezers valuable tools for probing critical biological interactions with positively charged amino acid side chains and cofactors. Molecular clips and tweezers can be employed for the complete inhibition of dehydrogenases. The clip extracts NAD(+) from its Rossman fold, while the tweezer complexes access strategic lysine residues around the active site. Our new enzyme inhibitors recognize the protein surface and thus offer additional targets for medicinal chemistry. Finally, the ability of molecular tweezers to cap critical lysine residues can be used to interfere with the pathology of protein misfolding diseases such as Alzheimers disease, because many of them involve noncovalent interactions with these critical residues during their early stages. When the key protein produces a β-sheet-rich nucleus, this structure undergoes spontaneous polymerization into highly toxic oligomers, ultimately leading to mature fibrils. The benzene-spaced phosphate tweezer forms a specific complex with lysine residues 16 and 28 in Aβ42 and thus prevents the formation of misfolded oligomers rich in β-sheets. This entirely new process-specific mechanism that prevents pathologic protein aggregation also operates in many other related amyloidogenic proteins.

Molecular clip and tweezer introduce new mechanisms of enzyme inhibition.

Artificial molecular clips and tweezers, designed for cofactor and amino acid recognition, are able to inhibit the enzymatic activity of alcohol dehydrogenase (ADH). IC50 values and kinetic investigations point to two different new mechanisms of interference with the NAD(+)-dependent oxidoreductase: While the clip seems to pull the cofactor out of its cleft, the tweezer docks onto lysine residues around the active site. Both modes of action can be reverted to some extent, by appropriate additives. However, while cofactor depletion by clip 1 was in part restored by subsequent NAD(+) addition, the tweezer (2) inhibition requires the competitive action of lysine derivatives. Lineweaver-Burk plots indicate a competitive mechanism for the clip, with respect to both substrate and cofactor, while the tweezer clearly follows a noncompetitive mechanism. Conformational analysis by CD spectroscopy demonstrates significant ADH denaturation in both cases. However, only the latter case (tweezer-lysine) is reversible, in full agreement with the above-detailed enzyme switch experiments. The complexes of ADH with clips or tweezer can be visualized in a nondenaturing gel electrophoresis, where the complexes migrate toward the anode, in contrast to the pure enzyme which approaches the cathode. Supramolecular chemistry has thus been employed as a means to control protein function with the specificity of artificial hosts opening new avenues for this endeavor.

Comparison of Three Amyloid Assembly Inhibitors: The Sugar scyllo- Inositol, the Polyphenol Epigallocatechin Gallate, and the Molecular Tweezer CLR01

Many compounds have been tested as inhibitors or modulators of amyloid β-protein (Aβ) assembly in hope that they would lead to effective, disease-modifying therapy for Alzheimers disease (AD). These compounds typically were either designed to break apart β-sheets or selected empirically. Two such compounds, the natural inositol derivative scyllo-inositol and the green-tea-derived flavonoid epigallocatechin gallate (EGCG), currently are in clinical trials. Similar to most of the compounds tested thus far, the mechanism of action of scyllo-inositol and EGCG is not understood. Recently, we discovered a novel family of assembly modulators, Lys-specific molecular tweezers, which act by binding specifically to Lys residues and modulate the self-assembly of amyloid proteins, including Aβ, into formation of nontoxic oligomers by a process-specific mechanism (Sinha, S., Lopes, D. H., Du, Z., Pang, E. S., Shanmugam, A., Lomakin, A., Talbiersky, P., Tennstaedt, A., McDaniel, K., Bakshi, R., Kuo, P. Y., Ehrmann, M., Benedek, G. B., Loo, J. A., Klarner, F. G., Schrader, T., Wang, C., and Bitan, G. (2011) Lysine-specific molecular tweezers are broad-spectrum inhibitors of assembly and toxicity of amyloid proteins. J. Am. Chem. Soc.133, 16958-16969). Here, we compared side-by-side the capability of scyllo-inositol, EGCG, and the molecular tweezer CLR01 to inhibit Aβ aggregation and toxicity. We found that EGCG and CLR01 had comparable activity whereas scyllo-inositol was a weaker inhibitor. Exploration of the binding of EGCG and CLR01 to Aβ using heteronuclear solution-state NMR showed that whereas CLR01 bound to the two Lys and single Arg residues in Aβ monomers, only weak, nonspecific binding was detected for EGCG, leaving the binding mode of the latter unresolved.

Modeling the Supramolecular Properties of Aliphatic‐Aromatic Hydrocarbons with Convex–Concave Topology

The molecular tweezers 1 a and 1 b have an electrostatic potential on the concave sides of the molecule which is surprisingly negative for hydrocarbons. According to semiempirical calculations this is a general phenomenon in nonconjugated π-electron systems with concave-convex topology, and it explains the receptor properties of 1 a and 1 b.

Molecular tweezers as synthetic receptors: molecular recognition of electron-deficient aromatic and aliphatic substrates.

The molecular tweezers 1 and 2 are selective receptors for electron-deficient aromatic and aliphatic substrates forming, for example, complexes with onium salts such as N-methylpyrazinium iodide, depending on the size and the electrostatic potential of the receptor cavity and the substrate.

Synthesis and supramolecular structures of molecular clips

The syntheses of the novel dimethylene-bridged clips I (n = 0, R = H, OAc, OH, OMe, OSO2CF3; n = 1, R = OAc) are reported. They selectively bind electron-deficient neutral and cationic arom. substrates comparable to tetramethylene-bridged tweezers. The geometry of the noncovalently bound complexes with I (n = 0; R = OAc, OH, OMe) as receptors, derived from single-crystal structure analyses, is, however, different from that of the tweezer complexes. In clip complexes the plane of the included arom. substrate mol. is oriented almost parallel to the naphthalene side-walls of the clip, whereas in the tweezer complex the substrate is oriented parallel to the central arene spacer-unit. 1,2,4,5-Benzenetetracarbonitrile (II) as substrate is placed inside the cavity of the hydroquinone clip (I; n = 0, R = OH) in soln. as well as in the cocrystal. In contrast, it was found for the cocrystal with the diacetate clip (I; n = 0, R = OAc) that II is placed between the naphthalene side-wall of two different clip mols. whereas in soln. II is included into the cavity of I (n = 0, R = OAc). Finally, II forms a 1:2 complex with dinaphthonorbornadiene in soln. as well as in the cryst. state. The findings reported here are instructive for the understanding of the weak noncovalent binding forces particularly the arene-arene interaction.

Molecular Clips with Extended Aromatic Sidewalls as Receptors for Electron-Acceptor Molecules. Synthesis and NMR, Photophysical, and Electrochemical Properties

We have synthesized molecular clips 1 comprising (i) two benzo[k]fluoranthene sidewalls and (ii) a dimethylene-connected benzene bridge that carries two acetoxy (1a), hydroxy (1b), or methoxy (1c) substituents in the para position. Their NMR spectra, single-crystal structures, and photophysical (fluorescence intensity, lifetime, depolarization) and electrochemical properties are discussed. For the purpose of comparison, similar compounds (2 and 3) containing only one benzo[k]fluoranthene unit have been prepared and studied. The strongly fluorescent clips 1 form stable complexes with electron-acceptor guests because of a highly negative electrostatic potential on the inner van der Waals surface of their cavity. The complexation constants in chloroform solution for a variety of guests, determined by NMR and fluorescence titration, are much larger than those of the corresponding anthracene and naphthalene clips (4 and 5), particularly in the case of extended aromatic guests. The effect of the substituents in the para position of the benzene spacer unit of clips 1 is discussed on the basis of the host-guest complex structures obtained by X-ray analysis and molecular mechanics simulations. In the case of 9-dicyanomethylene-2,4,7-trinitrofluorene (TNF) guest, complex formation with clip 1a causes dramatic changes in the photophysical and electrochemical properties: (i) a new charge-transfer band at 600 nm arises, (ii) a very efficient quenching of the strong benzo[k]fluoranthene fluorescence takes place, (iii) shifts of both the first oxidation (clip-centered) and reduction (TNF-centered) potentials are observed, and (iv) reversible disassembling of the complex can be obtained by electrochemical stimulation.

The Effect of Pressure on Organic Reactions

The utility of high pressure for the understanding of chemical reactions and its application in organic synthesis is shown for cycloadditions (inter- and intramolecular Diels-Alder reactions, 1,3-dipolar and [2+2] cycloadditions), cheletropic reactions and pericyclic rearrangements (Cope and Claisen rearrangements and electrocyclizations). The origin of the effect of pressure on chemical reactions is discussed. Especially, the change in the packing coefficient during cyclization of chains and the effect of electrostriction on reactions, in which charged species are generated, contribute substantially to a volume contraction leading to a powerful pressure-induced acceleration of such reactions. Finally, the effect of pressure on free-radical reactions (homolytic bond dissociations and quinone oxidations) is described.

Synthesis of sterically rigid macrocycles by the use of pressure-induced repetitive Diels-Alder reactions.

Synthesis of the syn-dimethanotetrahydroanthracene and -tetracene derivs. I (R1 = R2 = OMe, OH, OAc; R1 = Me, R2 = OH 2a-d) and II (R3 = OMe, OH 6a, b) are described. Highly stereoselective, pressure-induced repetitive Diels-Alder reactions of these bis-dienophiles with bis-diene III (7) lead to sterically rigid macrocycles having well defined cavities of different size.