Bernhard H. Monien

C-terminal peptides coassemble into Aβ42 oligomers and protect neurons against Aβ42-induced neurotoxicity

Alzheimers disease (AD) is an age-related disorder that threatens to become an epidemic as the world population ages. Neurotoxic oligomers of Aβ42 are believed to be the main cause of AD; therefore, disruption of Aβ oligomerization is a promising approach for developing therapeutics for AD. Formation of Aβ42 oligomers is mediated by intermolecular interactions in which the C terminus plays a central role. We hypothesized that peptides derived from the C terminus of Aβ42 may get incorporated into oligomers of Aβ42, disrupt their structure, and thereby inhibit their toxicity. We tested this hypothesis using Aβ fragments with the general formula Aβ(x−42) (x = 28–39). A cell viability screen identified Aβ(31–42) as the most potent inhibitor. In addition, the shortest peptide, Aβ(39–42), also had high activity. Both Aβ(31–42) and Aβ(39–42) inhibited Aβ-induced cell death and rescued disruption of synaptic activity by Aβ42 oligomers at micromolar concentrations. Biophysical characterization indicated that the action of these peptides likely involved stabilization of Aβ42 in nontoxic oligomers. Computer simulations suggested a mechanism by which the fragments coassembled with Aβ42 to form heterooligomers. Thus, Aβ(31–42) and Aβ(39–42) are leads for obtaining mechanism-based drugs for treatment of AD using a systematic structure–activity approach.

A novel allosteric pathway of thrombin inhibition: Exosite II mediated potent inhibition of thrombin by chemo-enzymatic, sulfated dehydropolymers of 4-hydroxycinnamic acids.

Thrombin and factor Xa, two important pro-coagulant proteinases, can be regulated through direct and indirect inhibition mechanisms. Recently, we designed sulfated dehydropolymers (DHPs) of 4-hydroxycinnamic acids that displayed interesting anticoagulant properties (Monien, B. H., Henry, B. L., Raghuraman, A., Hindle, M., and Desai, U. R. (2006) Bioorg. Med. Chem. 14, 7988–7998). To better understand their mechanism of action, we studied the direct inhibition of thrombin, factor Xa, factor IXa, and factor VIIa by CDSO3, FDSO3, and SDSO3, three analogs of sulfated DHPs. All three sulfated DHPs displayed a 2–3-fold preference for direct inhibition of thrombin over factor Xa, whereas this preference for inhibiting thrombin over factor IXa and factor VIIa increased to 17–300-fold, suggesting a high level of selectivity. Competitive binding studies with a thrombin-specific chromogenic substrate, a fluorescein-labeled hirudin peptide, bovine heparin, enoxaparin, and a heparin octasaccharide suggest that CDSO3 preferentially binds in or near anion-binding exosite II of thrombin. Studies of the hydrolysis of H-d-hexahydrotyrosol-Ala-Arg-p-nitroanilide indicate that CDSO3 inhibits thrombin through allosteric disruption of the catalytic apparatus, specifically through the catalytic step. Overall, designed sulfated DHPs appear to be the first molecules that bind primarily in the region defined by exosite II and allosterically induce thrombin inhibition. The molecules are radically different in structure from all the current clinically used anticoagulants and thus represent a novel class of potent dual thrombin and factor Xa inhibitors.

Structural Basis for Aβ1–42 Toxicity Inhibition by Aβ C-Terminal Fragments: Discrete Molecular Dynamics Study

Amyloid β-protein (Aβ) is central to the pathology of Alzheimers disease. Of the two predominant Aβ alloforms, Aβ(1-40) and Aβ(1-42), the latter forms more toxic oligomers. C-terminal fragments (CTFs) of Aβ were recently shown to inhibit Aβ(1-42) toxicity in vitro. Here, we studied Aβ(1-42) assembly in the presence of three effective CTF inhibitors and an ineffective fragment, Aβ(21-30). Using a discrete molecular dynamics approach that recently was shown to capture key differences between Aβ(1-40) and Aβ(1-42) oligomerization, we compared Aβ(1-42) oligomer formation in the absence and presence of CTFs or Aβ(21-30) and identified structural elements of Aβ(1-42) that correlated with Aβ(1-42) toxicity. CTFs co-assembled with Aβ(1-42) into large heterooligomers containing multiple Aβ(1-42) and inhibitor fragments. In contrast, Aβ(21-30) co-assembled with Aβ(1-42) into heterooligomers containing mostly a single Aβ(1-42) and multiple Aβ(21-30) fragments. The CTFs, but not Aβ(21-30), decreased the β-strand propensity of Aβ(1-42) in a concentration-dependent manner. CTFs and Aβ(21-30) had a high binding propensity to the hydrophobic regions of Aβ(1-42), but only CTFs were found to bind the Aβ(1-42) region A2-F4. Consequently, only CTFs but not Aβ(21-30) reduced the solvent accessibility of Aβ(1-42) in region D1-R5. The reduced solvent accessibility of Aβ(1-42) in the presence of CTFs was comparable to the solvent accessibility of Aβ(1-40) oligomers formed in the absence of Aβ fragments. These findings suggest that region D1-R5, which was more exposed to the solvent in Aβ(1-42) than in Aβ(1-40) oligomers, is involved in mediating Aβ(1-42) oligomer neurotoxicity.

Biophysical Characterization of Aβ42 C-Terminal Fragments: Inhibitors of Aβ42 Neurotoxicity

A key event in Alzheimers disease (AD) is age-dependent, brain accumulation of amyloid beta-protein (Abeta) leading to Abeta self-association into neurotoxic oligomers. Previously, we showed that Abeta oligomerization and neurotoxicity could be inhibited by C-terminal fragments (CTFs) of Abeta42. Because these CTFs are highly hydrophobic, we asked if they themselves aggregated and, if so, what parameters regulated their aggregation. To answer these questions, we investigated the dependence of CTF aqueous solubility, aggregation kinetics, and morphology on peptide length and sequence and the correlation between these characteristics and inhibition of Abeta42-induced toxicity. We found that CTFs up to 8 residues long were soluble at concentrations >100 microM and had a low propensity to aggregate. Longer CTFs were soluble at approximately 1-80 microM, and most, but not all, readily formed beta-sheet-rich fibrils. Comparison to Abeta40-derived CTFs showed that the C-terminal dipeptide I41-A42 strongly promoted aggregation. Aggregation propensity correlated with the previously reported tendency to form beta-hairpin conformation but not with inhibition of Abeta42-induced neurotoxicity. The data enhance our understanding of the physical characteristics that affect CTF activity and advance our ability to design, synthesize, and test future generations of inhibitors.

Mechanistic Investigation of the Inhibition of Aβ42 Assembly and Neurotoxicity by Aβ42 C-Terminal Fragments

Oligomeric forms of amyloid beta-protein (Abeta) are key neurotoxins in Alzheimers disease (AD). Previously, we found that C-terminal fragments (CTFs) of Abeta42 interfered with assembly of full-length Abeta42 and inhibited Abeta42-induced toxicity. To decipher the mechanism(s) by which CTFs affect Abeta42 assembly and neurotoxicity, here, we investigated the interaction between Abeta42 and CTFs using photoinduced cross-linking and dynamic light scattering. The results demonstrate that distinct parameters control CTF inhibition of Abeta42 assembly and Abeta42-induced toxicity. Inhibition of Abeta42-induced toxicity was found to correlate with stabilization of oligomers with a hydrodynamic radius (R(H)) of 8-12 nm and attenuation of formation of oligomers with an R(H) of 20-60 nm. In contrast, inhibition of Abeta42 paranucleus formation correlated with CTF solubility and the degree to which CTFs formed amyloid fibrils themselves but did not correlate with inhibition of Abeta42-induced toxicity. Our findings provide important insight into the mechanisms by which different CTFs inhibit the toxic effect of Abeta42 and suggest that stabilization of nontoxic Abeta42 oligomers is a promising strategy for designing inhibitors of Abeta42 neurotoxicity.

Early diagnostics and therapeutics for Alzheimer’s disease – how early can we get there?

Alzheimer’s disease (AD) is a major threat for the rapidly aging world population. AD is the leading cause of dementia and a major cause of death in developed countries. The disease puts a tremendous practical, emotional and financial burden on individuals and governments. Clinicians and researchers in the AD field face great challenges: the pathophysiological processes that cause AD are not well understood, definite diagnosis of AD requires autopsy, and therapeutic options are limited to treating the symptoms rather than the cause of the disease. Nevertheless, new insights into the earliest events that lead to development of AD increase hope that reliable diagnostics and efficacious therapies may emerge.

Synthesis and Purification of Highly Hydrophobic Peptides Derived from the C-Terminus of Amyloid β-Protein.

Some biotechnological inventions involve expensive, sophisticated machines. Others are relatively simple innovations that nevertheless address, and solve difficult problems. Synthesis and purification of highly hydrophobic peptides can be a difficult and challenging task, particularly when these peptides have low solubility in both aqueous and organic solvents. Here we describe the synthesis and purification of a series of peptides derived from the hydrophobic C-terminus of the 42-residue form of amyloid β-protein (Aβ42), a peptide believed to be the primary cause for Alzheimers disease (AD). The series of C-terminal fragments (CTFs) had the general formula Aβ(x-42), x=28-39, which potentially can be used as inhibitors of Aβ42 assembly and neurotoxicity. Synthesis and purification of peptides containing 8-residues or less were straightforward. However, HPLC purification of longer peptides was problematic and provided <1% yield in particularly difficult cases due to very poor solubility in the solvent systems used both in reverse- and in normal phase chromatography. Modification of the purification protocol using water precipitation followed by removal of scavengers by washing with diethyl ether circumvented the need for HPLC purification and provided these peptides with purity as high as HPLC-purified peptides and substantially increased yield.

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.