Pseudopeptide Amyloid Aggregation Inhibitors: In Silico, Single Molecule and Cell Viability Studies.

Neurodegeneration in Alzheimer's disease (AD) is defined by pathology featuring amyloid-ß (Aß) deposition in the brain. Aß monomers themselves are generally considered to be nontoxic, but misfold into ß-sheets and aggregate to form neurotoxic oligomers. One suggested strategy to treat AD is to prevent the formation of toxic oligomers. The SG inhibitors are a class of pseudopeptides designed and optimized using molecular dynamics (MD) simulations for affinity to Aß and experimentally validated for their ability to inhibit amyloid-amyloid binding using single molecule force spectroscopy (SMFS). In this work, we provide a review of our previous MD and SMFS studies of these inhibitors and present new cell viability studies that demonstrate their neuroprotective effects against Aß(1-42) oligomers using mouse hippocampal-derived HT22 cells. Two of the tested SG inhibitors, predicted to bind Aß in anti-parallel orientation, demonstrated neuroprotection against Aß(1-42). A third inhibitor, predicted to bind parallel to Aß, was not neuroprotective. Myristoylation of SG inhibitors, intended to enhance delivery across the blood-brain barrier (BBB), resulted in cytotoxicity. This is the first use of HT22 cells for the study of peptide aggregation inhibitors. Overall, this work will inform the future development of peptide aggregation inhibitors against Aß toxicity.

Exploring Amyloid-ß Dimer Structure Using Molecular Dynamics Simulations.

A major hallmark of Alzheimer's disease (AD) is the aggregation of amyloid-ß peptides in the brains of people afflicted by the disease. The exact pathway to this catastrophic event is unknown. In this work, a total of 9.5 µs molecular dynamics simulations have been performed to investigate the structure and dynamics of the smallest form of toxic Aß oligomers, i.e., the Aß dimers. This study suggests that specific hydrophobic regions are vital in the aggregation process. Different possible structures for Aß dimers are reported along with their relative binding affinity. These data may be used to design better Aß-aggregation inhibitors. The diversity of the dimer structures suggests several aggregation pathways.

Pseudopeptide Designed to Inhibit Oligomerization and Redox Chemistry in Alzheimer?s Disease.

Aggregation of amyloid beta peptide (A?) and inflammatory processes associated with the generation of superoxide, hydrogen peroxide, and hydroxyl radicals are responsible for neurotoxicity in Alzheimer?s disease. The latter are a result of the redox activity of copper-bound A? complexes with molecular oxygen. A ligand (PI1), previously designed to compete with A? for copper, and a pseudopeptide (SGC1) with a property of breaking the self-aggregation of A?, are attached to each other to form a new pseudopeptide (TGC1) in this study. Using a combination of density functional theory and molecular dynamics simulations, we show that TGC1 should have the ability to inhibit self-aggregation of A?, prevent the binding of copper to A?, and quench the redox activity of the copper. This trifunctional ligand is a good candidate for development into an anti-Alzheimer drug.

New Orbital Symmetry-Allowed Route for Cycloreversion of Silacyclobutane and Its Methyl Derivatives.

The [2+2] cycloreversion of silacyclobutane (SCB) and its two methyl-substituted derivatives, 1-methyl-1-silacyclobutane (MSCB) and 1,1-dimethyl-1-silacyclobutane (DMSCB), were studied using ab initio quantum chemistry calculations. The second-order Møller-Plesset (MP2) perturbation theory, complete active space self-consistent field (CASSCF), and coupled clusters methods were used to explore both the concerted and the stepwise cycloreversions of the three molecules. In addition to the orbital symmetry-forbidden supra-supra [2s+2s] transition state, a new orbital symmetry-allowed supra-antara [2s+2a] transition state was discovered for the concerted route for all three molecules. Both methyl substitution and temperature play a role in the kinetic competition between the [2s+2s] and [2s+2a] routes. At 0 and 298 K, the concerted [2s+2a] cycloreversion is kinetically more favorable than the [2s+2s] cycloreversion for SCB, but the opposite is true for MSCB and DMSCB. With increasing temperatures to above 600 and 1800 K, the [2s+2a] cycloreversion becomes more favorable for MSCB and DMSCB, respectively. The methyl substitutions on Si atoms also affect the stability of the diradical intermediate formed by Si-C bond rupture, leading to a less stable diradical with increasing methyl groups.

d-Amino Acid Pseudopeptides as Potential Amyloid-Beta Aggregation Inhibitors.

A causative factor for neurotoxicity associated with Alzheimer's disease is the aggregation of the amyloid-ß (Aß) peptide into soluble oligomers. Two all d-amino acid pseudo-peptides, SGB1 and SGD1, were designed to stop the aggregation. Molecular dynamics (MD) simulations have been carried out to study the interaction of the pseudo-peptides with both Aß13⁻23 (the core recognition site of Aß) and full-length Aß1⁻42. Umbrella sampling MD calculations have been used to estimate the free energy of binding, ∆G, of these peptides to Aß13⁻23. The highest ∆Gbinding is found for SGB1. Each of the pseudo-peptides was also docked to Aß1⁻42 and subjected up to seven microseconds of all atom molecular dynamics simulations. The resulting structures lend insight into how the dynamics of Aß1⁻42 are altered by complexation with the pseudo-peptides and confirmed that SGB1 may be a better candidate for developing into a drug to prevent Alzheimer's disease.

ß- N-Methylamino-l-alanine (BMAA) Not Involved in Alzheimer's Disease.

ß- N-Methylamino-l-alanine (BMAA) is a neurotoxic agent implicated in ALS as well as Parkinson's and Alzheimer's diseases. It is produced by blue-green algae and could find its way via fish and seafood into the human food supply. Isolation from biological samples yields the compound in monomeric and protein-bound form. It has been suggested that the protein-bound fraction may result from genetic misincorporation into proteins in place of serine. Concomitant misfolding of the mutated proteins may be responsible for the neurological diseases. Recent reports that contradict the misincorporation theory leave unresolved the nature of the protein-bound form of BMAA. We have found from quantum mechanical calculations on model systems that it is possible to bind BMAA with high affinity in a noncovalent fashion to proteins. Because of our interest in Alzheimer's disease, molecular dynamics simulations were applied to search for such binding between BMAA and the ß-amyloid peptide and to discover whether replacement of either of its two serine residues could affect its aggregation into neurotoxic oligomers. No stable noncovalently bound complex was found, and it was concluded that incorporation of BMAA in place of serine would not alter the conformational dynamics of the ß-amyloid peptide.

Computational modelling of the redistribution of copper isotopes by proteins in the liver.

Changes in the stable isotope composition of copper in blood serum as a result of biological processes in the liver were quantified as coupled equilibrium fractionation processes. The model used calculated reduced partition function ratios corresponding to interactions involving individual proteins using Density Functional Theory. This quantified the effect that each process had on the redistribution of copper isotopes in the liver. It was not possible to calculate the reduced partition function of CTR1 as a high resolution crystal structure of its copper binding domains are unavailable at the time of writing, and an optimization process was used to estimate the reduced partition function of CTR1 and constrain the possible isotopic fractionation associated with interactions involving CTR1 independent of direct DFT calculations and assumptions of its structure. The exchange of copper between ceruloplasmin and ATP7B has the most significant impact on the copper isotopic composition of blood serum. The model calculation for the isotopic composition of ceruloplasmin and albumin are δ65Cu = (-0.54 ± 0.10)‰ and δ65Cu = (0.08 ± 0.25)‰ respectively, assuming that serum is 90% ceruloplasmin and 10% albumin using a measured δ65Cu of serum of (0.52 ± 0.08)‰. The model also predicts that the isotopic composition of the tri-nuclear binding motif of ceruloplasmin may be relatively depleted in the lighter isotope of Cu compared to the other copper binding sites by as much as -1.08 ± 0.45‰.

Copper(I) Chelators for Alzheimer's Disease.

A component of the neurotoxicity of the beta amyloid peptide (Aß) of Alzheimer's disease is its ability to generate superoxide, hydrogen peroxide, and hydroxyl radicals by reaction of its reduced copper complex Aß/Cu+ with molecular oxygen. The objective of the present work was to devise compounds, L, that could remove Cu+ from Aß/Cu+, with the property that L/Cu+ itself would not be capable of reducing O2 or hydrogen peroxide. We show by density functional calculations that several pincer-type compounds with two imidazole rings and a sulfur or nitrogen have the desired combination of Cu+ binding affinity and Cu2+ reduction potential.

Testing synthetic amyloid-ß aggregation inhibitor using single molecule atomic force spectroscopy.

Alzheimer's disease is a neurodegenerative disease with no known cure and few effective treatment options. The principal neurotoxic agent is an oligomeric form of the amyloid-ß peptide and one of the treatment options currently being studied is the inhibition of amyloid aggregation. In this work, we test a novel pseudopeptidic aggregation inhibitor designated as SG1. SG1 has been designed to bind at the amyloid-ß self-recognition site and prevent amyloid-ß from misfolding into ß sheet. We used atomic force spectroscopy, a nanoscale measurement technique, to quantify the binding forces between two single amyloid peptide molecules. For the first time, we demonstrate that single molecule atomic force spectroscopy can be used to assess the effectiveness of amyloid aggregation inhibitors by measuring the experimental yield of binding and can potentially be used as a screening technique for quick testing of efficacy of inhibitor drugs for amyloid aggregation.

Fe(III)-Heme Complexes with the Amyloid Beta Peptide of Alzheimer's Disease: QM/MM Investigations of Binding and Redox Properties of Heme Bound to the His Residues of Aß(1-42).

Pursuant to our previous paper [J. Chem. Theory Comput. 2012, 8, 5150-5158], the structures of complexes between Aß(1-42) and ferriheme (Fe(III)-heme-H2O) were determined by application of Amber and ONIOM(B3LYP/6-31G(d):Amber) methodology. Attachment at each of the three His residues was investigated. As well as direct bonding of the iron to the His residue, bonding is augmented by formation of secondary salt bridges between the carboxylate groups of the heme and positively charged residues of Aß (at His13, by Lys16 and the N-terminus; at His14, by Lys16; at His6, by Arg5). The results indicate a slight preference for His13 followed by His6 and His14, with the lowest 10 structures lying within 30 kJ mol(-1) of each other. The absolute binding affinities are predicted to be approximately 30-40 kJ mol(-1). Standard reduction potentials (E°) are calculated for various Fe(III)/Fe(II) couples. Regardless of the point of attachment of the heme, E° values are approximately -0.6 V relative to the standard hydrogen electrode.

NMR and computational studies of the configurational properties of spirodioxyselenuranes. Are dynamic exchange processes or temperature-dependent chemical shifts involved?

Spirodioxyselenurane 4a and several substituted analogs revealed unexpected (1)H NMR behavior. The diastereotopic methylene hydrogens of 4a appeared as an AB quartet at low temperature that coalesced to a singlet upon warming to 267 K, suggesting a dynamic exchange process with a relatively low activation energy. However, DFT computational investigations indicated high activation energies for exchange via inversion through the selenium center and for various pseudorotation processes. Moreover, the NMR behavior was unaffected by the presence of water or acid catalysts, thereby ruling out reversible Se-O or benzylic C-O cleavage as possible stereomutation pathways. Remarkably, when 4a was heated beyond 342 K, the singlet was transformed into a new AB quartet. Further computations indicated that a temperature dependence of the chemical shifts of the diastereotopic protons results in convergence upon heating, followed by crossover and divergence at still higher temperatures. The NMR behavior is therefore not due to dynamic exchange processes, but rather to temperature dependence of the chemical shifts of the diastereotopic hydrogens, which are coincidentally equivalent at intermediate temperatures. These results suggest the general need for caution in ascribing the coalescence of variable-temperature NMR signals of diastereotopic protons to dynamic exchange processes that could instead be due to temperature-dependent chemical shifts and highlight the importance of corroborating postulated exchange processes through additional computations or experiments wherever possible.

The Binding of Fe(II)-Heme to the Amyloid Beta Peptide of Alzheimer's Disease: QM/MM Investigations.

The structures of complexes between Aß(1-42) and ferroheme (Fe(II)-heme) were determined by application of Amber and ONIOM(B3LYP/6-31G(d):Amber) methodology. Attachment at each of the three His residues was investigated. In each case, direct bonding of the iron to the His residue is augmented by the formation of secondary salt bridges between the carboxylate groups of the heme and positively charged residues of Aß (at His13, by Lys16 and the N-terminus; at His14, by both Lys16 and Lys28; at His6, by Arg5) or by H-bonding and hydrophobic interactions (at His6, by Asp7 or Phe20). The results indicate a slight preference for His13 followed by His6 and His14, with the lowest eight structures lying within 36 kJ mol(-1) of each other. The methodology is not precise enough to permit a definitive statement as to the relative stabilities, nor to the absolute binding affinities, which are predicted to be less than 70 kJ mol(-1). The results bear on the question of how heme and copper may bind simultaneously to Aß. They confirm that the reduced species can bind independently, Cu(+) at His13-His14 and Fe(II)-heme at His6.

The structures and stabilities of the complexes of biologically available ligands with Fe(II) porphine: an Ab initio study.

Fe(II) porphine complexes were investigated at the "MP2/LB"//B3LYP/SB level of theory where SB and LB denote the small and large basis sets, 6-31+G(d) and 6-311+G(2df,2p), respectively. Solvation effects due to water and benzene were approximated by the IEFPCM procedure. Ligands included H2O, Cl-, and OH- and Im(imidazole), CH3NH2, (CH3)2S, CH3CO2-, CH3 S-, and CH3PhO- as models of the side chains of His, Lys, Met, Asp/Glu, Cys, and Tyr residues, respectively. Fe(II)porphine, 2, and the complexes 2(H2O), 2(Im), 2(CH3)2S), 2(CH3NH2), and 2(H2O)2 have triplet ground states. All pentacoordinated complexes of 2 with negatively charged ligands have high spin quintet ground states, while all hexacoordinated complexes with at least one Im ligand have low spin singlet ground states. With the exception of 2(Im)2 and 2(Im)((CH3)2S), no hexacoordinated complexes are stable in water or benzene.Redox properties are sensitive to the nature of the environment and the ligand(s)attached to the iron center. With the exception of the parent system, 2+/2, all complexes are predicted to have a negative reduction potential relative to the standard hydrogen electrode in water. With neutral ligand(s),the reduction potential is higher in the nonpolar environment. The opposite is true with negatively charged ligands. The redox activity of cytochromes, peroxidases, and catalases is discussed in the context of the model parent systems.

On the involvement of copper binding to the N-terminus of the amyloid Beta Peptide of Alzheimer's disease: a computational study on model systems.

Density functional and second order Moller-Plesset perturbation theoretical methods, coupled with a polarizable continuum model of water, were applied to determine the structures, binding affinities, and reduction potentials of Cu(II) and Cu(I) bound to models of the Asp1, Ala2, His6, and His13His14 regions of the amyloid beta peptide of Alzheimer's disease. The results indicate that the N-terminal Asp binds to Cu(II) together with His6 and either His13 or His14 to form the lower pH Component I of Aß. Component II of Aß is the complex between Cu(II) and His6, His13, and His14, to which an amide O (of Ala2) is also coordinated. Asp1 does not bind to Cu(II) if three His residues are attached nor to any Cu(I) species to which one or more His residues are bound. The most stable Cu(I) species is one in which Cu(I) bridges the N(δ) of His13 and His14 in a linear fashion. Cu(I) binds more strongly to Aß than does Cu(II). The computed reduction potential that closely matches the experimental value for Cu(II)/Aß corresponds to reduction of Component II (without Ala2) to the Cu(I) complex after endergonic attachment of His6.

Structures and stabilities of Fe2+/3+ complexes relevant to Alzheimer's disease: an ab initio study.

Iron is one of the most abundant metals found in senile plaques of post mortem patients with Alzheimer's disease. However, the interaction mode between iron ions and ß-amyloid peptide as well as their precise affinity is unknown. In this study we apply ab initio computational methodology to calculate binding energies of Fe(2+/3+) with the His13-His14 sequence of Aß, as well as other important ligands such as His6 and Tyr10. Calculations were carried out at the "MP2/6-311+G(2df,2p)"//B3LYP/6-31+G(d) level of theory and solvent effects included by the IEFPCM procedure. Several reaction paths for the binding of imidazole, phenol, and the His13-His14 fragment (modeled by N-(2-(1H-imidazol-4-yl)ethyl)-3-(1H-imidazol-4-yl)propanamide) were sequentially explored. The results show that the most stable complexes containing His13-His14 and phenolate of Tyr10 are the pentacoordinated [Fe(2+)(O-HisHis)(PhO(-))(H(2)O)](+) and [Fe(3+)(N-HisHis)(PhO(-))(H(2)O)](+) compounds and that simultaneous coordination of tyrosine and His13-His14 to Fe(2+/3+) is thermodynamically favorable in water at physiological pH. Computed Raman spectra confirm the conclusion obtained by Miura et al. ( Biochemistry 2000 , 39 , 7024 ) that tyrosine is coordinated to Fe(3+) but do not exclude coordination of imidazoles. Finally, calculations of standard reduction potentials indicate that phenol coordination reduces the redox activity of the iron/Aß complexes.

The structures and stabilities of the complexes of biologically available ligands with Fe(III)-porphine: an ab initio study.

Fe(III) porphine complexes and a limited number of Fe(II) porphine complexes were investigated at the 'MP2/LB'//B3LYP/SB level of theory, where SB and LB represent small and large basis sets, 6-31+G(d) and 6-311+G(2df,2p), respectively. Solvation effects were incorporated by the IEFPCM procedure. Most of the ligands whereby the heme prosthetic group is bound in biological systems were modeled in the study. These include H(2)O, Im (imidazole), CH(3)NH(2), CH(3)CO(2)(-), CH(3)S(-), CH(3)PhO(-), OH(-), and Cl(-). Fe(III) porphine, 2(+), and the pentacoordinated complexes, 2(+)(Im), 2(+)(CH(3)NH(2)), and 2(+)(H(2)O), have quartet ground states. The pentacoordinated complexes with negatively charged ligands all have high spin hextet ground states. All of the hexacoordinated complexes have low spin doublet ground states, with the exception of 2(+)(H(2)O)(2) and 2(+)(H(2)O)(Im) which have intermediate spin quartet ground states. None of the pentacoordinated complexes, 2(+)(OH(-)), 2(+)(CH(3)PhO(-)), and 2(+)(CH(3)S(-)), are predicted to form stable hexacoordinated complexes in water with any of the ligands of the present study. The most stable species in water is 2(+)(OH(-)). The hydroxide may be displaced by CH(3)PhOH and CH(3)SH at physiological pH, and by Cl(-), CH(3)CO(2)(-), and Im under acidic conditions, but not by CH(3)NH(3)(+). The relevance of the present results for the pH-dependent transitions of cytochrome c and the fragments, NAcMP8, and NAcMP11, the resting state of cytochrome P450, and the bonding interactions between heme and Aß, is discussed.

Synthesis and cyclooxygenase inhibitory activities of some N-acylhydrazone derivatives of isoxazolo[4,5-d]pyridazin-4(5H)-ones.

In this study, new isoxazolo[4,5-d]pyridazin-4(5H)-one derivatives having an N-acylhydrazone moiety were synthesized. The compounds were tested for their COX inhibitory activities using NS-398 and indomethacine as reference compounds. Although the compounds had an inhibitory profile against both COX-1 and COX-2, most were found to be more selective against COX-2 by a small percentage of inhibition, at the concentration of 50 microM. Docking studies were done to understand the interactions of the tested compounds with the active site of COX-2. It was observed that the compounds fit into, and interacted with, the hydrophobic parts which are common in the active pocket of COX-1 and COX-2 enzymes but could not fit to the area which is specific for COX-2 enzyme.

The chemistry of Alzheimer's disease.

The chemistry of Alzheimer's disease is largely centred on the amyloid beta-peptide, its formation, structure, and interactions with metals, membranes, proteins and other species. This critical review summarizes the current state of knowledge (252 references).

Can copper binding to the prion protein generate a misfolded form of the protein?

The native prion protein (PrP) has a two domain structure, with a globular folded alpha-helical C-terminal domain and a flexible extended N-terminal region. The latter can selectively bind Cu(2+) via four His residues in the octarepeat (OR) region, as well as two sites (His96 and His111) outside this region. In the disease state, the folded C-terminal domain of PrP undergoes a conformational change, forming amorphous aggregates high in beta-sheet content. Cu(2+) bound to the ORs can be redox active and has been shown to induce cleavage within the OR region, a process requiring conserved Trp residues. Using computational modeling, we have observed that electron transfer from Trp residues to copper can be favorable. These models also reveal that an indole-based radical cation or Cu(+) can initiate reactions leading to protein backbone cleavage. We have also demonstrated, by molecular dynamics simulations, that Cu(2+) binding to the His96 and His111 residues in the remaining PrP N-terminal fragment can induce localized beta-sheet structure, allowing us to suggest a potential mechanism for the initiation of beta-sheet misfolding in the C-terminal domain by Cu(2+).

Mechanism of hydrogen peroxide production by copper-bound amyloid beta peptide: a theoretical study.

The amyloid beta peptide (Abeta) of Alzheimer's disease evolves hydrogen peroxide in vitro in the presence of Cu(II), external reducing agents, and molecular oxygen, without producing detectable amounts of the one-electron reduced intermediate, superoxide, O(2)(-*). The mechanism of this process was examined by ab initio computational chemistry techniques in systems that model the binding of Cu(II) to the His13His14 fragment of Abeta. The catalytic cycle begins with the reduction of the most stable Cu(II) complex to the most stable Cu(I) complex. This Cu(I) complex forms a Cu(II)-like adduct with (3)O(2) that cannot dissociate in water to yield O(2)(-*). However, it can be reduced by proton-coupled electron transfer to an adduct between HOO(-) and the Cu(II)-like complex, which in turn can be protonated. The protonated complex decomposes to yield H(2)O(2) by an associative-dissociative mechanism, thus completing the cycle.