New strategy for the generation of specific D-peptide amyloid inhibitors

The conversion of a soluble protein into β-sheet-rich oligomeric structures and further fiber formation are critical steps in the pathogenesis of the group of human diseases known as amyloidoses. Drugs that interfere with this process may thus be able to prevent and/or cure these diseases. Recent results have shown that short amino acid stretches can provide most of the driving force needed to trigger amyloid formation of a protein. These evidence suggest that compounds that specifically bind to peptides synthesized upon the sequence of such amyloidogenic protein stretches might also be able to inhibit amyloid formation of the corresponding full-length protein and, likely, amyloid-induced cytotoxicity as well. Here we present a general strategy to obtain d-peptides that specifically interact with protein amyloid stretches. The screening of a d-peptide combinatorial library for inhibitors of an amyloidogenic peptide designed de novo has allowed us to extract a set of empirical rules for the design of d-peptide inhibitors of any six-residue amyloidogenic stretch. d-peptides generated on these bases prevent amyloid formation and disassemble preformed fibrils of different amyloid hexapeptides identified in human amyloid proteins. In addition, they are also specific for their target sequence. The d-peptide designed here for the Alzheimer's Aβ_1-42 peptide not only inhibits and disassembles amyloid material but also reduces Aβ_1-42 amyloid-induced cytotoxicity in cell culture.     

Neurotoxicity of Alzheimer's disease A�� peptides is induced by small changes in the A��42 to A��40 ratio

The amyloid peptides Aβ₄₀ and Aβ₄₂ of Alzheimer's disease are thought to contribute differentially to the disease process. Although Aβ₄₂ seems more pathogenic than Aβ₄₀, the reason for this is not well understood. We show here that small alterations in the Aβ₄₂:Aβ₄₀ ratio dramatically affect the biophysical and biological properties of the Aβ mixtures reflected in their aggregation kinetics, the morphology of the resulting amyloid fibrils and synaptic function tested in vitro and in vivo. A minor increase in the Aβ₄₂:Aβ₄₀ ratio stabilizes toxic oligomeric species with intermediate conformations. The initial toxic impact of these Aβ species is synaptic in nature, but this can spread into the cells leading to neuronal cell death. The fact that the relative ratio of Aβ peptides is more crucial than the absolute amounts of peptides for the induction of neurotoxic conformations has important implications for anti‐amyloid therapy. Our work also suggests the dynamic nature of the equilibrium between toxic and non‐toxic intermediates.     

Size-dependent neurotoxicity of β-amyloid oligomers

The link between the size of soluble amyloid β (Aβ) oligomers and their toxicity to rat cerebellar granule cells (CGC) was investigated. Variation in conditions during in vitro oligomerization of Aβ_1-42 resulted in peptide assemblies with different particle size as measured by atomic force microscopy and confirmed by dynamic light scattering and fluorescence correlation spectroscopy. Small oligomers of Aβ_1-42 with a mean particle z-height of 1-2 nm exhibited propensity to bind to phospholipid vesicles and they were the most toxic species that induced rapid neuronal necrosis at submicromolar concentrations whereas the bigger aggregates (z-height above 4-5 nm) did not bind vesicles and did not cause detectable neuronal death. A similar neurotoxic pattern was also observed in primary cultures of cortex neurons whereas Aβ₁₋₄₂ oligomers, monomers and fibrils were non-toxic to glial cells in CGC cultures or macrophage J774 cells. However, both oligomeric forms of Aβ_1-42 induced reduction of neuronal cell densities in the CGC cultures.     

Pretreatment of chemically-synthesized Aβ42 affects its biological activity in yeast

The tendency of amyloid β (Aβ₄₂) peptide to misfold and aggregate into insoluble amyloid fibrils in Alzheimer's disease (AD) has been well documented. Accumulation of Aβ₄₂ fibrils has been correlated with abnormal apoptosis and unscheduled cell division which can also trigger the death of neuronal cells, while oligomers can also exhibit similar activities. While investigations using chemically-synthesized Aβ₄₂ peptide have become common practice, there appear to be differences in outcomes from different preparations. In order to resolve this inconsistency, we report 2 separate methods of preparing chemically-synthesized Aβ₄₂ and we examined their effects in yeast. Hexafluoroisopropanol pretreatment caused toxicity while, ammonium hydroxide treated Aβ₄₂ induced cell proliferation in both C. glabrata and S. cerevisiae. The hexafluoroisopropanol prepared Aβ₄₂ had greater tendency to form amyloid on yeast cells as determined by thioflavin T staining followed by flow cytometry and microscopy. Both quiescent and non-quiescent cells were analyzed by these methods of peptide preparation. Non-quiescent cells were susceptible to the toxicity of Aβ₄₂ compared with quiescent cells (p < 0.005). These data explain the discrepancy in the previous publications about the effects of chemically-synthesized Aβ₄₂ on yeast cells. The effect of Aβ₄₂ on yeast cells was independent of the size of the peptide aggregates. However, the Aβ₄₂ pretreatment determined whether the molecular conformation of peptide resulted in proliferation or toxicity in yeast based assays.     

Amyloid β (1-42) Folding Multiplicity and Single-Molecule Binding Behavior Studied with STM

The fine folding and assembling characteristics of amyloid β (Aβ) peptides are important to pharmaceutical studies of drug molecules and to the pathological analysis of neurodegenerative disorders such as Alzheimer's disease at the molecular level. Here we present observations of the multiple folding characteristics of amyloid peptide Aβ₄₂ lamellae using scanning tunneling microscopy. Molecularly resolved core regions of Aβ₄₂ hairpins and unfolded peptide assembly structures are identified. The parallel assembling characteristics of Aβ₄₂ hairpins can be confirmed in the study. In addition, single-molecule binding characteristics of Congo red and thioflavin T have been shown to bind at the groove regions of peptide assemblies. This study demonstrates a complementary venue for studying molecular heterogeneity of peptide assemblies, as well as the binding characteristics of molecular modulators.     

Intrinsic Determinants of Neurotoxic Aggregate Formation by the Amyloid β Peptide

The extent to which proteins aggregate into distinct structures ranging from prefibrillar oligomers to amyloid fibrils is key to the pathogenesis of many age-related degenerative diseases. We describe here for the Alzheimer's disease-related amyloid β peptide (Aβ) an investigation of the sequence-based determinants of the balance between the formation of prefibrillar aggregates and amyloid fibrils. We show that by introducing single-point mutations, it is possible to convert the normally harmless Aβ₄₀ peptide into a pathogenic species by increasing its relative propensity to form prefibrillar but not fibrillar aggregates, and, conversely, to abolish the pathogenicity of the highly neurotoxic E22G Aβ₄₂ peptide by reducing its relative propensity to form prefibrillar species rather than mature fibrillar ones. This observation can be rationalized by the demonstration that whereas regions of the sequence of high aggregation propensity dominate the overall tendency to aggregate, regions with low intrinsic aggregation propensities exert significant control over the balance of the prefibrillar and fibrillar species formed, and therefore play a major role in determining the neurotoxicity of the Aβ peptide.     

Effect of agitation on the peptide fibrillization: Alzheimer's amyloid‐β peptide 1‐42 but not amylin and insulin fibrils can grow under quiescent conditions

Many peptides and proteins can form fibrillar aggregates in vitro, but only a limited number of them are forming pathological amyloid structures in vivo. We studied the fibrillization of four peptides – Alzheimer's amyloid‐β (Aβ) 1‐40 and 1‐42, amylin and insulin. In all cases, intensive mechanical agitation of the solution initiated fast fibrillization. However, when the mixing was stopped during the fibril growth phase, the fibrillization of amylin and insulin was practically stopped, and the rate for Aβ₄₀ substantially decreased, whereas the fibrillization of Aβ₄₂ peptide continued to proceed with almost the same rate as in the agitated conditions. The reason for the different sensitivity of the in vitro fibrillization of these peptides towards agitation in the fibril growth phase remains elusive. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Axon degeneration is key component of neuronal death in amyloid-β toxicity

Depending upon the stimulus, neuronal cell death can either be triggered from the cell body (soma) or the axon. We investigated the origin of the degeneration signal in amyloid β (Aβ) induced neuronal cell death in cultured in vitro hippocampal neurons. We discovered that Aβ_1–42 toxicity-induced axon degeneration precedes cell death in hippocampal neurons. Overexpression of Bcl-xl inhibited both axonal and cell body degeneration in the Aβ-42 treated neurons. Nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) blocks axon degeneration in a variety of paradigms, but it cannot block neuronal cell body death. Therefore, if the neuronal death signals in Aβ_1–42 toxicity originate from degenerating axons, we should be able to block neuronal death by inhibiting axon degeneration. To explore this possibility we over-expressed Nmnat1 in hippocampal neurons. We found that inhibition of axon degeneration in Aβ_1–42 treated neurons prevented neuronal cell death. Thus, we conclude that axon degeneration is the key component of Aβ_1–42 induced neuronal degeneration, and therapies targeting axonal protection can be important in finding a treatment for Alzheimer’s disease.     

Interaction of dipeptydil peptidase IV with amyloid peptides

The aggregates of amyloid beta peptides (Aβs) are regarded as one of the main pathological hallmarks of Alzheimer’s disease (AD). An imbalance between the rates of synthesis and clearance of Aβs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аβs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aβ40 and Aβ42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aβ40 and Aβ42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aβ40 (K_m = 37.5 μM, k_cat/K_m = 1.7 × 10³ M⁻¹sec⁻¹) and Aβ42 (K_m = 138.4 μM, k_cat/K_m = 1.90 × 10² M⁻¹sec⁻¹). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20 mM phosphate buffer, pH 7.4, 37 °C by 50–80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30–40%. Conclusion: for the first time it was shown that the Aβ40 and Aβ42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates.     

Hexafluoroisopropanol induces self‐assembly of β‐amyloid peptides into highly ordered nanostructures

Deposition of insoluble fibrillar aggregates of β‐amyloid (Aβ) peptides in the brain is a hallmark of Alzheimer's disease. Apart from forming fibrils, these peptides also exist as soluble aggregates. Fibrillar and a variety of nonfibrillar aggregates of Aβ have also been obtained in vitro. Hexafluoroisopropanol (HFIP) has been widely used to dissolve Aβ and other amyloidogenic peptides. In this study, we show that the dissolution of Aβ40, 42, and 43 in HFIP followed by drying results in highly ordered aggregates. Although α‐helical conformation is observed, it is not stable for prolonged periods. Drying after prolonged incubation of Aβ40, 42, and 43 peptides in HFIP leads to structural transition from α‐helical to β‐conformation. The peptides form short fibrous aggregates that further assemble giving rise to highly ordered ring‐like structures. Aβ16–22, a highly amyloidogenic peptide stretch from Aβ, also formed very similar rings when dissolved in HFIP and dried. HFIP could not induce α‐helical conformation in Aβ16–22, and rings were obtained from freshly dissolved peptide. The rings formed by Aβ40, 42, 43, and Aβ16–22 are composed of the peptides in β‐conformation and cause enhancement in thioflavin T fluorescence, suggesting that the molecular architecture of these structures is amyloid‐like. Our results clearly indicate that dissolution of Aβ40, 42 and 43 and the amyloidogenic fragment Aβ16–22 in HFIP results in the formation of annular amyloid‐like structures. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Influence of Residue 22 on the Folding, Aggregation Profile, and Toxicity of the Alzheimer's Amyloid β Peptide

Several biophysical techniques have been used to determine differences in the aggregation profile (i.e., the secondary structure, aggregation propensity, dynamics, and morphology of amyloid structures) and the effects on cell viability of three variants of the amyloid β peptide involved in Alzheimer's disease. We focused our study on the Glu²² residue, comparing the effects of freshly prepared samples and samples aged for at least 20 days. In the aged samples, a high propensity for aggregation and β-sheet secondary structure appears when residue 22 is capable of establishing polar (Glu²² in wild-type) or hydrophobic (Val²² in E22V) interactions. The Arctic variant (E22G) presents a mixture of mostly disordered and α-helix structures (with low β-sheet contribution). Analysis of transmission electron micrographs and atomic force microscopy images of the peptide variants after aging showed significant quantitative and qualitative differences in the morphology of the formed aggregates. The effect on human neuroblastoma cells of these Aβ_12-28 variants does not correlate with the amount of β-sheet of the aggregates. In samples allowed to age, the native sequence was found to have an insignificant effect on cell viability, whereas the Arctic variant (E22G), the E22V variant, and the slightly-aggregating control (F19G-F20G) had more prominent effects.     

Dynamics of Inter-Molecular Interactions Between Single Aβ42 Oligomeric and Aggregate Species by High-Speed Atomic Force Microscopy

Within the amyloid hypothesis in Alzheimer's disease, current focus has shifted to earlier stages of amyloid beta (Aβ) peptide assembly, involving soluble oligomers and smaller aggregates, which are more toxic to cells compared to their morphological distinct fibril forms. Critical to the Aβ field is unlocking the molecular-level kinetic pathways of oligomerization, leading to the culprit subset or specific species of Aβ oligomer populations responsible for the disease etiology. Here, we apply high-speed atomic force microscopy to enable direct visualization of dynamic interactions between single Aβ₄₂ oligomers and aggregate forms, with combined nanometre structural and millisecond temporal resolution in liquid. Analysis of dimensions revealed up to three main Aβ₄₂ species distributions, in addition to the appearance of monomers that showed fast surface diffusion compared to the larger Aβ₄₂ species. Significantly, we devised a new single-molecule analysis based on image contrast in high-speed atomic force microscopy movies to quantify rate determining kinetic constants for interactions between the different Aβ₄₂ species. The findings revealed that smaller Aβ₄₂ species show an exponential decay of lifetime distribution, indicating that all molecules undergo the same process with a single well-defined energy barrier. In contrast, larger aggregates show randomized lifetimes, indicating a distribution of interactions energies/barriers that must be overcome in order to dissociate. We interpret the latter as being due to “permissive” binding, arising from different conformation states of the aggregates, along with a variety of accessible interacting groups. Inevitably, this may lead to the formation of different complexes or alloforms, which is known to contribute to difficulties in identifying Aβ oligomer toxicity and has implications for mechanisms underlying neuronal death accompanying Alzheimer's disease.     

A modified protocol to prepare seed-free starting solutions of amyloid-β (Aβ)1–40 and Aβ1–42 from the corresponding depsipeptides

Preparing reliable, seed-free stock solutions of the highly amyloidogenic peptides amyloid-β (Aβ) is difficult. Besides the formation of aggregates during synthesis and storage, dissolution of the peptide is a critical step because vortexing can induce aggregation. To overcome this, synthesis of the more water-soluble depsi-Aβ_1–42 peptide, from which the native sequence is easily obtained, has been suggested. We further refined this technique, including a cutoff filtration step and switching the depsipeptide in basic conditions, to stabilize the formed native peptide. The obtained solutions of native Aβ_1–40 and Aβ_1–42 peptides were homogeneous and aggregate free, as indicated by thioflavin T and circular dichroism analysis.     

Cultured cell and transgenic mouse models for tau pathology linked to β-amyloid

The two histopathological signatures of Alzheimer's disease (AD) are amyloid plaques and neurofibrillary tangles, prompting speculation that a causal relationship exists between the respective building blocks of these abnormal brain structures: the β-amyloid peptides (Aβ) and the neuron-enriched microtubule-associated protein called tau. Transgenic mouse models have provided in vivo evidence for such connections, and cultured cell models have allowed tightly controlled, systematic manipulation of conditions that influence links between Aβ and tau. The emerging evidence supports the view that amyloid pathology lies upstream of tau pathology in a pathway whose details remain largely mysterious. In this communication, we review and discuss published work about the Aβ–tau connection. In addition, we present some of our own previously unpublished data on the effects of exogenous Aβ on primary brain cultures that contain both neurons and glial cells. We report here that continuous exposure to 5 μM non-fibrillar Aβ40 or Aβ42 kills primary brain cells by apoptosis within 2–3 weeks, Aβ42 is more toxic and selective for neurons than Aβ40, and Aβ42, but not Aβ40, induces a transient increase in neurons that are positive for the AD-like PHF1 epitope. These findings demonstrate the greater potency of Aβ42 than Aβ40 at inducing tau pathology and programmed cell death, and corroborate and extend reports that tau-containing cells are more sensitive to Aβ peptides than cells that lack or express low levels of tau.     

Intracellular selection of peptide inhibitors that target disulphide-bridged Aβ42 oligomers

The β-amyloid (Aβ) peptide aggregates into a number of soluble and insoluble forms, with soluble oligomers thought to be the primary factor implicated in Alzheimer''s disease pathology. As a result, a wide range of potential aggregation inhibitors have been developed. However, in addition to problems with solubility and protease susceptibility, many have inadvertently raised the concentration of these soluble neurotoxic species. Sandberg et al. previously reported a β-hairpin stabilized variant of Aβ₄₂ that results from an intramolecular disulphide bridge (A21C/A31C; Aβ_42cc), which generates highly toxic oligomeric species incapable of converting into mature fibrils. Using an intracellular protein-fragment complementation (PCA) approach, we have screened peptide libraries using E. coli that harbor an oxidizing environment to permit cytoplasmic disulphide bond formation. Peptides designed to target either the first or second β-strand have been demonstrated to bind to Aβ_42cc, lower amyloid cytotoxicity, and confer bacterial cell survival. Peptides have consequently been tested using wild-type Aβ₄₂ via ThT binding assays, circular dichroism, MTT cytotoxicity assays, fluorescence microscopy, and atomic force microscopy. Results demonstrate that amyloid-PCA selected peptides function by both removing amyloid oligomers as well as inhibiting their formation. These data further support the use of semirational design combined with intracellular PCA methodology to develop Aβ antagonists as candidates for modification into drugs capable of slowing or even preventing the onset of AD.     

Amyloid oligomer formation probed by water proton magnetic resonance spectroscopy

Formation of amyloid oligomers, the most toxic species of amyloids in degenerative diseases, is critically coupled to the interplay with surrounding water. The hydrophobic force driving the oligomerization causes water removal from interfaces, changing the surface-hydration properties. Here, we show that such effects alter the magnetic relaxation response of local water in ways that may enable oligomer detection. By using water proton magnetic resonance spectroscopy, we measured significantly longer transverse magnetic relaxation (T₂) times in mixtures of serum and amyloidogenic Aβ_1-42 peptides versus similar concentration solutions of serum and nonamyloidogenic scrambled Aβ_42-1 peptides. Immunochemistry with oligomer-specific antibodies, electron microscopy and computer simulations demonstrated that the hyperintense magnetic signal correlates with Aβ_1-42 oligomerization. Finding early biophysical markers of the oligomerization process is crucial for guiding the development of new noninvasive imaging techniques, enabling timely diagnosis of amyloid-related diseases and pharmacological intervention.     

Selective cytotoxicity of intracellular amyloid beta peptide1-42 through p53 and Bax in cultured primary human neurons

Extracellular amyloid beta peptides (Abetas) have long been thought to be a primary cause of Alzheimer's disease (AD). Now, detection of intracellular neuronal Abeta1--42 accumulation before extracellular Abeta deposits questions the relevance of intracellular peptides in AD. In the present study, we directly address whether intracellular Abeta is toxic to human neurons. Microinjections of Abeta1--42 peptide or a cDNA-expressing cytosolic Abeta1--42 rapidly induces cell death of primary human neurons. In contrast, Abeta1--40, Abeta40--1, or Abeta42--1 peptides, and cDNAs expressing cytosolic Abeta1--40 or secreted Abeta1--42 and Abeta1--40, are not toxic. As little as a 1-pM concentration or 1500 molecules/cell of Abeta1--42 peptides is neurotoxic. The nonfibrillized and fibrillized Abeta1--42 peptides are equally toxic. In contrast, Abeta1--42 peptides are not toxic to human primary astrocytes, neuronal, and nonneuronal cell lines. Inhibition of de novo protein synthesis protects against Abeta1--42 toxicity, indicating that programmed cell death is involved. Bcl-2, Bax-neutralizing antibodies, cDNA expression of a p53R273H dominant negative mutant, and caspase inhibitors prevent Abeta1--42-mediated human neuronal cell death. Taken together, our data directly demonstrate that intracellular Abeta1--42 is selectively cytotoxic to human neurons through the p53--Bax cell death pathway.     

A synthetic peptide corresponding to a region of the human pericentriolar material 1 (PCM‐1) protein binds β‐amyloid (Aβ1‐42) oligomers

We have recently reported that a ~19‐kDa polypeptide, rPK‐4, is a protein kinase Cs inhibitor that is 89% homologous to the 1171–1323 amino acid region of the 228‐kDa human pericentriolar material‐1 (PCM‐1) protein (Chakravarthy et al. 2012). We have now discovered that rPK‐4 binds oligomeric amyloid‐β peptide (Aβ)_1‐42 with high affinity. Most importantly, a PCM‐1‐selective antibody co‐precipitated Aβ and amyloid β precursor protein (AβPP) from cerebral cortices and hippocampi from AD (Alzheimer's disease) transgenic mice that produce human AβPP and Aβ_1‐42, suggesting that PCM‐1 may interact with amyloid precursor protein/Aβ in vivo. We have identified rPK‐4′s Aβ‐binding domain using a set of overlapping synthetic peptides. We have found with ELISA, dot‐blot, and polyacrylamide gel electrophoresis techniques that a ~ 5 kDa synthetic peptide, amyloid binding peptide (ABP)‐p4‐5 binds Aβ_1‐42 at nM levels. Most importantly, ABP‐p4‐5, like rPK‐4, appears to preferentially bind Aβ_1‐42 oligomers, believed to be the toxic AD‐drivers. As expected from these observations, ABP‐p4‐5 prevented Aβ_1‐42 from killing human SH‐SY5Y neuroblastoma cells via apoptosis. These findings indicate that ABP‐p4‐5 is a possible candidate therapeutic for AD.     

Expression in drosophila of tandem amyloid β peptides provides insights into links between aggregation and neurotoxicity

The generation and subsequent aggregation of amyloid β (Aβ) peptides play a crucial initiating role in the pathogenesis of Alzheimer disease (AD). The two main isoforms of these peptides have 40 (Aβ(40)) or 42 residues (Aβ(42)), the latter having a higher propensity to aggregate in vitro and being the main component of the plaques observed in vivo in AD patients. We have designed a series of tandem dimeric constructs of these Aβ peptides to probe the manner in which changes in the aggregation kinetics of Aβ affect its deposition and toxicity in a Drosophila melanogaster model system. The levels of insoluble aggregates were found to be substantially elevated in flies expressing the tandem constructs of both Aβ(40) and Aβ(42) compared with the equivalent monomeric peptides, consistent with the higher effective concentration, and hence increased aggregation rate, of the peptides in the tandem repeat. A unique feature of the Aβ(42) constructs, however, is the appearance of high levels of soluble oligomeric aggregates and a corresponding dramatic increase in their in vivo toxicity. The toxic nature of the Aβ(42) peptide in vivo can therefore be attributed to the higher kinetic stability of the oligomeric intermediate states that it populates relative to those of Aβ(40) rather than simply to its higher rate of aggregation.     

Binding Modes of Phthalocyanines to Amyloid β Peptide and Their Effects on Amyloid Fibril Formation

The inherent tendency of proteins to convert from their native states into amyloid aggregates is associated with a range of human disorders, including Alzheimer’s and Parkinson’s diseases. In that sense, the use of small molecules as probes for the structural and toxic mechanism related to amyloid aggregation has become an active area of research. Compared with other compounds, the structural and molecular basis behind the inhibitory interaction of phthalocyanine tetrasulfonate (PcTS) with proteins such as αS and tau has been well established, contributing to a better understanding of the amyloid aggregation process in these proteins. We present here the structural characterization of the binding of PcTS and its Cu(II) and Zn(II)-loaded forms to the amyloid β-peptide (Aβ) and the impact of these interactions on the peptide amyloid fibril assembly. Elucidation of the PcTS binding modes to Aβ₄₀ revealed the involvement of specific aromatic and hydrophobic interactions in the formation of the Aβ₄₀-PcTS complex, ascribed to a binding mode in which the planarity and hydrophobicity of the aromatic ring system in the phthalocyanine act as main structural determinants for the interaction. Our results demonstrated that formation of the Aβ₄₀-PcTS complex does not interfere with the progression of the peptide toward the formation of amyloid fibrils. On the other hand, conjugation of Zn(II) but not Cu(II) at the center of the PcTS macrocyclic ring modified substantially the binding profile of this phthalocyanine to Aβ₄₀ and became crucial to reverse the effects of metal-free PcTS on the fibril assembly of the peptide. Overall, our results provide a firm basis to understand the structural rules directing phthalocyanine-protein interactions and their implications on the amyloid fibril assembly of the target proteins; in particular, our results contradict the hypothesis that PcTS might have similar mechanisms of action in slowing the formation of a variety of pathological aggregates.