Molecular tweezers with varying anions: a comparative study.

Selective binding of the phosphate-substituted molecular tweezer 1a to protein lysine residues was suggested to explain the inhibition of certain enzymes and the aberrant aggregation of amyloid petide Aß42 or α-synuclein, which are assumed to be responsible for Alzheimer's and Parkinson's disease, respectively. In this work we systematically investigated the binding of four water-soluble tweezers 1a-d (substituted by phosphate, methanephosphonate, sulfate, or O-methylenecarboxylate groups) to amino acids and peptides containing lysine or arginine residues by using fluorescence spectroscopy, NMR spectroscopy, and isothermal titration calorimetry (ITC). The comparison of the experimental results with theoretical data obtained by a combination of QM/MM and ab initio(1)H NMR shift calculations provides clear evidence that the tweezers 1a-c bind the amino acid or peptide guest molecules by threading the lysine or arginine side chain through the tweezers' cavity, whereas in the case of 1d the guest molecule is preferentially positioned outside the tweezer's cavity. Attractive ionic, CH-π, and hydrophobic interactions are here the major binding forces. The combination of experiment and theory provides deep insight into the host-guest binding modes, a prerequisite to understanding the exciting influence of these tweezers on the aggregation of proteins and the activity of enzymes.

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 ß-protein (Aß) 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 Aß at the earliest stages of assembly. The resulting complexes were indistinguishable in size and morphology from Aß 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.

A mechanism of efficient G6PD inhibition by a molecular clip.

Triple duty: A synthetic molecular clip traps nicotinamide adenine dinucleotide phosphate (NADP(+); see picture) as well as occupying both the cofactor- and the substrate-binding site in glucose-6-phosphate (G6P) dehydrogenase. This combination of two inhibition mechanisms makes the clip highly effective and selective for this enzyme over other dehydrogenases.

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.

A new pyrene-based fluorescent probe for the determination of critical micelle concentrations.

A new pyrene-based fluorescent probe for the determination of critical micelle concentrations (CMC) is described. The title compound 1 is obtained in five steps, starting from pyrene. Fluorescence spectroscopic properties of 1 are studied in homogeneous organic solvents and aqueous micellar solutions. In a wide range of organic solvents, probe 1 exhibits a characteristic monomer emission of the pyrene fluorophore, with three distinct peak maxima at 382, 404, and 425 nm. The spectra change dramatically in aqueous solution, where no monomer emission of the pyrene fluorophore is detected. Instead, only strong excimer fluorescence with a broad, red-shifted emission band at lambda(max) = 465 nm is observed. In micellar aqueous solution, a superposition of the monomer and excimer emission is found. The appearance of the monomer emission in micellar solution can be explained on the basis of solubilization of 1 by the surfactant micelles. The ratio of the monomer to excimer fluorescence intensities of 1 is highly sensitive to changes in surfactant concentration. This renders 1 a versatile and sensitive probe molecule for studying the micellization of ionic and nonionic surfactants. For a representative selection of common surfactants, the critical micelle concentrations in aqueous solution are determined, showing excellent agreement with established literature data.