Thomas Schrader

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.

Functional Synthetic Receptors

Preface. List of Contributors. 1 Artif icial (Pseudo)peptides for Molecular Recognition and Catalysis (L. J. Prins, P. Scrimin). 1.1 Introduction. 1.2 Recognition of Biological Targets by Pseudo-peptides. 1.3 Synthetic (Pseudo)peptide-based Supermolecules: From Structure to Function. 1.4 Combinatorial Selection of Functional (Pseudo)peptides. 1.5 Conclusions. References. 2 Carbohydrate Receptors (A. P. Davis, T. D. James). 2.1 Introduction. 2.2 Carbohydrate Receptors Employing Noncovalent Interactions. 2.3 Receptors Employing B-O Bond Formation. References. 3 Ammonium, Amidinium, Guanidinium, and Pyridinium Cations (T. Schrader, M. Maue). 3.1 Introduction. 3.2 Ammonium Cations. 3.3 Amidinium Cations. 3.4 Guanidinium Cations. 3.5 Pyridinium Cations. 3.6 Conclusions and Outlook. References. 4 Artif icial Pyrrole-based Anion Receptors (W.-S. Cho, J. L. Sessler). 4.1 Introduction. 4.2 Anions in Biological Systems. 4.3 Cationic Pyrrole-based Receptors. 4.4 Neutral Pyrrole-based Anion Receptors. 4.5 Anion Carriers in Transport Applications. 4.6 Anion Sensing. 4.7 Guanidinium-based Anion Receptors. 4.8 Amide-based Anion Receptors. 4.9 Urea-based Anion Receptors. 4.10 Conclusions. Acknowledgment. References. 5 Molecular Containers in Action (D. M. Rudkevich). 5.1 Introduction. 5.2 Variety of Molecular Containers. 5.3 Chemistry Inside Capsules. 5.4 Storage of Information Inside Capsules. 5.5 Materials and Sensors by Encapsulation. 5.6 Biologically Relevant Encapsulation. 5.7 Concluding Remarks. Acknowledgment. References. 6 Formation and Recognition Properties of Dynamic Combinatorial Libraries (A. D. Hamilton, D. M. Tagore, K. I. Sprinz). 6.1 Introduction. 6.2 Covalent Interactions Used in DCC Design. 6.3 Noncovalent Interactions Used in DCC Design. 6.4 Conformational/Configurational Isomerization. 6.5 Receptor-based Screening, Selection, and Amplification. 6.6 Conclusions. Acknowledgment. References. 7 Synthetic Molecular Machines (E. R. Kay, D. A. Leigh). 7.1 Introduction. 7.2 Controlling Conformational Changes. 7.3 Controlling Configurational Changes. 7.4 Controlling Motion in Supramolecular Systems. 7.5 Controlling Motion in Interlocked Systems. 7.6 From Laboratory to Technology: Toward Useful Molecular Machines. 7.7 Summary and Outlook. References. 8 Replicable Nanoscaffolded Multifunctionality - A Chemical Perspective (W.-M. Pankau, S. Antsypovich, L. Eckardt, J. Stankiewicz, S. Monninghoff, J. Zimmermann, M. Radeva, G. von Kiedrowski). Abstract. 8.1 Introduction. 8.2 A Manifesto for Nanorobot Implementation. 8.3 Conclusion. Acknowledgment. References. Index.

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.

Unexpected enhancement of enantioselectivity in copper(II) catalyzed conjugate addition of diethylzinc to cyclic enones with novel TADDOL phosphorus amidite ligands

Abstract The copper(II) catalyzed enantioselective 1,4-addition reactions of diethylzinc to cyclic enones in the presence of novel phosphorus amidite ligands, easily prepared from α,α,α′,α′-tetraphenyl-2,2′-dimethyl-1,3-dioxolane-4,5-dimethanol (TADDOL) derivatives, resulted in e.e.s up to 71% for cyclohexenone and up to 62% for cyclopentenone. A remarkable enhancement of enantioselectivity was observed upon the addition of powdered molecular sieves to the reaction mixture.

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.

Structure of the protected dipeptide Ac-Val-Phe-OMe in the gas phase: Towards a β-sheet model system

In this paper we report on the structure of the isolated dipeptide Ac–Val–Phe–OMe (Val=valine, Phe=phenylalanine) which is protected at the terminal positions by introducing an acetyl and a methyl group. Both resonant two-photon ionization (R2PI) and IR/R2PI spectroscopy are applied. This is the first application of IR/R2PI spectroscopy to a dipeptide. Both the region of the C–H and N–H stretching vibrations as well as the region of the C=O stretching vibrations are investigated. The chosen dipeptide exhibits only one prominent conformer in the gas phase containing a “linear” non-hydrogen-bonded structure which is an ideal candidate for a β-sheet model.

Investigation of Secondary Structure Elements by IR/UV Double Resonance Spectroscopy : Analysis of an Isolated β-Sheet Model System

An isolated beta-sheet model system is investigated in a molecular beam experiment by means of mass- and isomer-selective IR/R2PI double resonance spectroscopy as well as ab initio and DFT calculations. As the exclusive intermolecular assembly, a beta-sheet motif is formed by spontaneous dimerization of two isolated peptide molecules. This secondary structure is produced from the tripeptide model Ac-Val-Tyr(Me)-NHMe without any further environment to form the binding motif which is analyzed by both the characteristic amide A and I vibrations. The experimental and theoretical investigations yield the assignment to an antiparallel beta-sheet model. The result of this detailed spectroscopic analysis on an isolated beta-sheet model indicates that there are intrinsic properties of a beta-sheet structure which can be formed without a solvent or a peptidic environment.

Rational design of β-sheet ligands against Aβ42-induced toxicity.

A β-sheet-binding scaffold was equipped with long-range chemical groups for tertiary contacts toward specific regions of the Alzheimers Aβ fibril. The new constructs contain a trimeric aminopyrazole carboxylic acid, elongated with a C-terminal binding site, whose influence on the aggregation behavior of the Aβ(42) peptide was studied. MD simulations after trimer docking to the anchor point (F19/F20) suggest distinct groups of complex structures each of which featured additional specific interactions with characteristic Aβ regions. Members of each group also displayed a characteristic pattern in their antiaggregational behavior toward Aβ. Specifically, remote lipophilic moieties such as a dodecyl, cyclohexyl, or LPFFD fragment can form dispersive interactions with the nonpolar cluster of amino acids between I31 and V36. They were shown to strongly reduce Thioflavine T (ThT) fluorescence and protect cells from Aβ lesions (MTT viability assays). Surprisingly, very thick fibrils and a high β-sheet content were detected in transmission electron microscopy (TEM) and CD spectroscopic experiments. On the other hand, distant single or multiple lysines which interact with the ladder of stacked E22 residues found in Aβ fibrils completely dissolve existing β-sheets (ThT, CD) and lead to unstructured, nontoxic material (TEM, MTT). Finally, the triethyleneglycol spacer between heterocyclic β-sheet ligand and appendix was found to play an active role in destabilizing the turn of the U-shaped protofilament. Fluorescence correlation spectroscopy (FCS) and sedimentation velocity analysis (SVA) provided experimental evidence for some smaller benign aggregates of very thin, delicate structure (TEM, MTT). A detailed investigation by dynamic light scattering (DLS) and other methods proved that none of the new ligands acts as a colloid. The evolving picture for the disaggregation mechanism by these new hybrid ligands implies transformation of well-ordered fibrils into less structured aggregates with a high molecular weight. In the few cases where fibrillar components remain, these display a significantly altered morphology and have lost their acute cellular toxicity.

Molecular Basis for Preventing α-Synuclein Aggregation by a Molecular Tweezer

Background: The molecular tweezer, CLR01, binds to Lys and prevents aggregation of α-synuclein. Results: CLR01 binds directly to monomeric α-synuclein near the N terminus and changes the charge distribution in the sequence, swelling the chain, and increasing the protein reconfiguration rate. Conclusion: Aggregation is inhibited by making the protein more diffusive. Significance: The most effective aggregation inhibitors may change monomer dynamics rather than structure. Recent work on α-synuclein has shown that aggregation is controlled kinetically by the rate of reconfiguration of the unstructured chain, such that the faster the reconfiguration, the slower the aggregation. In this work we investigate this relationship by examining α-synuclein in the presence of a small molecular tweezer, CLR01, which binds selectively to Lys side chains. We find strong binding to multiple Lys within the chain as measured by fluorescence and mass-spectrometry and a linear increase in the reconfiguration rate with concentration of the inhibitor. Top-down mass-spectrometric analysis shows that the main binding of CLR01 to α-synuclein occurs at the N-terminal Lys-10/Lys-12. Photo-induced cross-linking of unmodified proteins (PICUP) analysis shows that under the conditions used for the fluorescence analysis, α-synuclein is predominantly monomeric. The results can be successfully modeled using a kinetic scheme in which two aggregation-prone monomers can form an encounter complex that leads to further oligomerization but can also dissociate back to monomers if the reconfiguration rate is sufficiently high. Taken together, the data provide important insights into the preferred binding site of CLR01 on α-synuclein and the mechanism by which the molecular tweezer prevents self-assembly into neurotoxic aggregates by α-synuclein and presumably other amyloidogenic proteins.