Dahabada H. J. Lopes

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.

A key role for lysine residues in amyloid β-protein folding, assembly, and toxicity.

A combination of hydrophobic and electrostatic interactions is important in initiating the aberrant self-assembly process that leads to formation of toxic oligomers and aggregates by multiple disease-related proteins, including amyloid β-protein (Aβ), whose self-assembly is believed to initiate brain pathogenesis in Alzheimers disease. Lys residues play key roles in this process and participate in both types of interaction. They also are the target of our recently reported molecular tweezer inhibitors. To obtain further insight into the role of the two Lys residues in Aβ assembly and toxicity, here we substituted each by Ala in both Aβ40 and Aβ42 and studied the impact of the substitution on Aβ oligomerization, aggregation, and toxicity. Our data show that each substitution has a major impact on Aβ assembly and toxicity, with significant differences depending on peptide length (40 versus 42 amino acids) and the position of the substitution. In particular, Lys16→Ala substitution dramatically reduces Aβ toxicity. The data support the use of compounds targeting Lys residues specifically as inhibitors of Aβ toxicity and suggest that exploring the role of Lys residues in other disease-related amyloidogenic proteins may help understanding the mechanisms of aggregation and toxicity of these proteins.

Induction of methionine-sulfoxide reductases protects neurons from amyloid β-protein insults in vitro and in vivo.

Self-assembly of amyloid β-protein (Aβ) into toxic oligomers and fibrillar polymers is believed to cause Alzheimers disease (AD). In the AD brain, a high percentage of Aβ contains Met-sulfoxide at position 35, though the role this modification plays in AD is not clear. Oxidation of Met(35) to sulfoxide has been reported to decrease the extent of Aβ assembly and neurotoxicity, whereas surprisingly, oxidation of Met(35) to sulfone yields a toxicity similar to that of unoxidized Aβ. We hypothesized that the lower toxicity of Aβ-sulfoxide might result not only from structural alteration of the C-terminal region but also from activation of methionine-sulfoxide reductase (Msr), an important component of the cellular antioxidant system. Supporting this hypothesis, we found that the low toxicity of Aβ-sulfoxide correlated with induction of Msr activity. In agreement with these observations, in MsrA(-/-) mice the difference in toxicity between native Aβ and Aβ-sulfoxide was essentially eliminated. Subsequently, we found that treatment with N-acetyl-Met-sulfoxide could induce Msr activity and protect neuronal cells from Aβ toxicity. In addition, we measured Msr activity in a double-transgenic mouse model of AD and found that it was increased significantly relative to that of nontransgenic mice. Immunization with a novel Met-sulfoxide-rich antigen for 6 months led to antibody production, decreased Msr activity, and lowered hippocampal plaque burden. The data suggest an important neuroprotective role for the Msr system in the AD brain, which may lead to development of new therapeutic approaches for AD.

Design of β-Amyloid Aggregation Inhibitors from a Predicted Structural Motif

Drug design studies targeting one of the primary toxic agents in Alzheimers disease, soluble oligomers of amyloid β-protein (Aβ), have been complicated by the rapid, heterogeneous aggregation of Aβ and the resulting difficulty to structurally characterize the peptide. To address this, we have developed [Nle(35), D-Pro(37)]Aβ(42), a substituted peptide inspired from molecular dynamics simulations which forms structures stable enough to be analyzed by NMR. We report herein that [Nle(35), D-Pro(37)]Aβ(42) stabilizes the trimer and prevents mature fibril and β-sheet formation. Further, [Nle(35), D-Pro(37)]Aβ(42) interacts with WT Aβ(42) and reduces aggregation levels and fibril formation in mixtures. Using ligand-based drug design based on [Nle(35), D-Pro(37)]Aβ(42), a lead compound was identified with effects on inhibition similar to the peptide. The ability of [Nle(35), D-Pro(37)]Aβ(42) and the compound to inhibit the aggregation of Aβ(42) provides a novel tool to study the structure of Aβ oligomers. More broadly, our data demonstrate how molecular dynamics simulation can guide experiment for further research into AD.

Molecular tweezers inhibit islet amyloid polypeptide assembly and toxicity by a new mechanism.

In type-2 diabetes (T2D), islet amyloid polypeptide (IAPP) self-associates into toxic assemblies causing islet β-cell death. Therefore, preventing IAPP toxicity is a promising therapeutic strategy for T2D. The molecular tweezer CLR01 is a supramolecular tool for selective complexation of K residues in (poly)peptides. Surprisingly, it inhibits IAPP aggregation at substoichiometric concentrations even though IAPP has only one K residue at position 1, whereas efficient inhibition of IAPP toxicity requires excess CLR01. The basis for this peculiar behavior is not clear. Here, a combination of biochemical, biophysical, spectroscopic, and computational methods reveals a detailed mechanistic picture of the unique dual inhibition mechanism for CLR01. At low concentrations, CLR01 binds to K1, presumably nucleating nonamyloidogenic, yet toxic, structures, whereas excess CLR01 binds also to R11, leading to nontoxic structures. Encouragingly, the CLR01 concentrations needed for inhibition of IAPP toxicity are safe in vivo, supporting its development toward disease-modifying therapy for T2D.

C-Terminal Tetrapeptides Inhibit Aβ42-Induced Neurotoxicity Primarily through Specific Interaction at the N-Terminus of Aβ42

Inhibition of amyloid β-protein (Aβ)-induced toxicity is a promising therapeutic strategy for Alzheimers disease (AD). Previously, we reported that the C-terminal tetrapeptide Aβ(39-42) is a potent inhibitor of neurotoxicity caused by Aβ42, the form of Aβ most closely associated with AD. Here, initial structure-activity relationship studies identified key structural requirements, including chirality, side-chain structure, and a free N-terminus, which control Aβ(39-42) inhibitory activity. To elucidate the binding site(s) of Aβ(39-42) on Aβ42, we used intrinsic tyrosine (Y) fluorescence and solution-state NMR. The data suggest that Aβ(39-42) binds at several sites, of which the predominant one is located in the N-terminus of Aβ42, in agreement with recent modeling predictions. Thus, despite the small size of Aβ(39-42) and the hydrophobic, aliphatic nature of all four side-chains, the interaction of Aβ(39-42) with Aβ42 is controlled by specific intermolecular contacts requiring a combination of hydrophobic and electrostatic interactions and a particular stereochemistry.

Tranilast Binds to Aβ Monomers and Promotes Aβ Fibrillation

The antiallergy and potential anticancer drug tranilast has been patented for treating Alzheimers disease (AD), in which amyloid β-protein (Aβ) plays a key pathogenic role. We used solution NMR to determine that tranilast binds to Aβ40 monomers with ∼300 μM affinity. Remarkably, tranilast increases Aβ40 fibrillation more than 20-fold in the thioflavin T assay at a 1:1 molar ratio, as well as significantly reducing the lag time. Tranilast likely promotes fibrillation by shifting Aβ monomer conformations to those capable of seed formation and fibril elongation. Molecular docking results qualitatively agree with NMR chemical shift perturbation, which together indicate that hydrophobic interactions are the major driving force of the Aβ-tranilast interaction. These data suggest that AD may be a potential complication for tranilast usage in elderly patients.

A Two-Step Strategy for Structure–Activity Relationship Studies of N-Methylated Aβ42 C-Terminal Fragments as Aβ42 Toxicity Inhibitors

Neurotoxic Aβ42 oligomers are believed to be the main cause of Alzheimer’s disease. Previously, we found that the C‐terminal fragments (CTFs), Aβ(30–42) and Aβ(31–42) were the most potent inhibitors of Aβ42 oligomerization and toxicity in a series of Aβ(x–42) peptides (x=28–39). Therefore, we chose these peptides as leads for further development. These CTFs are short (12–13 amino acids) hydrophobic peptides with limited aqueous solubility. Our first attempt to attach hydrophilic groups to the N terminus resulted in toxic peptides. Therefore, we next incorporated N‐methyl amino acids, which are known to increase the solubility of such peptides by disrupting the β‐sheet formation. Focusing on Aβ(31–42), we used a two‐step N‐methyl amino acid substitution strategy to study the structural factors controlling inhibition of Aβ42‐induced toxicity. First, each residue was substituted by N‐Me‐alanine (N‐Me‐A). In the next step, in positions where substitution produced a significant effect, we restored the original side chain. This strategy allowed exploring the role of both side chain structure and N‐Me substitution in inhibitory activity. We found that the introduction of an N‐Me amino acid was an effective way to increase both the aqueous solubility and the inhibitory activity of Aβ(31–42). In particular, N‐Me amino acid substitution at position 9 or 11 increased the inhibitory activity relative to the parent peptide. The data suggest that inhibition of Aβ42 toxicity by short peptides is highly structure‐specific, providing a basis for the design of new peptidomimetic inhibitors with improved activity, physicochemical properties, and metabolic stability.

Cytotoxic Mechanisms of Islet Amyloid Polypeptide in the Pathogenesis of Type-2 Diabetes Mellitus (T2DM)

Diabetes mellitus (DM) is a chronic syndrome that occurs due to loss of the insulin-producing β-cells in the islets of Langerhans of the pancreas by a β-cell-specific autoimmune process, leading to insulin deficiency (type-1 DM, T1DM) or when insufficient amounts of insulin are produced by β-cells, or when resistance to the action of insulin occurs in different tissues (type-2 DM, T2DM). Several mechanisms may contribute to the progressive β-cell failure in T2DM, including loss of β-cell mass, β-cell exhaustion, and the cytotoxic effects of elevated glucose and lipid levels. Another hallmark of T2DM is the accumulation of β-cell-produced amylin, also called islet amyloid polypeptide (IAPP), which forms amyloid that is present in approximately 95% of T2DM patients.

Structural insight into the mechanism of beta-amyloid toxicity and its inhibition by small molecules

Background: Amyloid b-protein (Ab) neurotoxicity is central in Alzheimer’s disease (AD) pathogenesis. The mechanisms by which Ab causes toxicity and the details of how small molecule modulators inhibit toxicity are open questions. Our recent discovery that Lys-specific molecular tweezers are effective inhibitors of Ab toxicity provides clues for answering these questions.Methods: The toxicity and self-assembly of Ab analogues containing Lysy’Ala substitutions was studied in vitro. Inhibition of Abinduced toxicity by the molecular tweezer CLR01, the sugar derivative scyllo-inositol, and the green-tea polyphenol EGCG was compared in cell culture. The interaction of CLR01 and EGCG with Ab was compared using 2D, heteronuclear, solution-state NMR. Results: Lys16 was found to be a major mediator of Ab toxicity, whereas Lys28 plays a key role in Ab assembly. CLR01 and EGCG are effective inhibitors of Ab oligomerization and toxicity, whereas scyllo-inositol is a substantially weaker inhibitor. CLR01 binds Ab already at the monomer state at defined positions, whereas EGCG binds Abmonomers weakly and non-specifically.Conclusions: The Lys residues in Ab are important for the peptide’s assembly and toxicity and therefore are attractive targets for development of small-molecule inhibitors of Ab-induced toxicity. Understanding the mechanism by which inhibitors and modulators work may facilitate future drug development.