RESUMO

A library of structurally related coumarins was generated through synthesis reactions and chemical modification reactions to obtain derivatives with antiproliferative activity both in vivo and in vitro. Out of a total of 35 structurally related coumarin derivatives, seven of them showed inhibitory activity in in vitro tests against Taq DNA polymerase with IC50 values lower than 250 µM. The derivatives 4-(chloromethyl)-5,7-dihydroxy-2H-chromen-2-one (2d) and 4-((acetylthio)methyl)-2-oxo-2H-chromen-7-yl acetate (3c) showed the most promising anti-polymerase activity with IC50 values of 20.7 ± 2.10 and 48.25 ± 1.20 µM, respectively. Assays with tumor cell lines (HEK 293 and HCT-116) were carried out, and the derivative 4-(chloromethyl)-7,8-dihydroxy-2H-chromen-2-one (2c) was the most promising, with an IC50 value of 8.47 µM and a selectivity index of 1.87. In addition, the derivatives were evaluated against Saccharomyces cerevisiae strains that report about common modes of actions, including DNA damage, that are expected for agents that cause replicative stress. The coumarin derivatives 7-(2-(oxiran-2-yl)ethoxy)-2H-chromen-2-one (5b) and 7-(3-(oxiran-2-yl)propoxy)-2H-chromen-2-one (5c) caused DNA damage in S. cerevisiae. The O-alkenylepoxy group stands out as that with the most important functionality within this family of 35 derivatives, presenting a very good profile as an antiproliferative scaffold. Finally, the in vitro antiretroviral capacity was tested through RT-PCR assays. Derivative 5c showed inhibitory activity below 150 µM with an IC50 value of 134.22 ± 2.37 µM, highlighting the O-butylepoxy group as the functionalization responsible for the activity.

RESUMO

Alzheimer's disease (AD) is related to the presence of extracellular aggregated amyloid-ß peptide (Aß), which binds copper(II) with high affinity in its N-terminal region. In this sense, two new 1-methylimidazole-containing N-acylhydrazonic metallophores, namely, X1TMP and X1Benz, were synthesized as hydrochlorides and characterized. The compound X1TMP contains the 3,4,5-trimethoxybenzoyl moiety present in the structure of mescaline, a natural hallucinogenic protoalkaloid that occurs in some species of cacti. Single crystals of X1Benz, the unsubstituted derivative of X1TMP, were obtained. The experimental partition coefficients of both compounds were determined, as well as their apparent affinity for Cu2+ in aqueous solution. Ascorbate consumption assays showed that these N-acylhydrazones are able to lessen the production of ROS by the Cu(Aß)-system, and a short-time scale aggregation study, measured through turbidity and confirmed by TEM images, revealed their capacity in preventing Aß fibrillation at equimolar conditions in the presence and absence of copper. 1H15N HSQC NMR experiments demonstrated a direct interaction between Aß and X1Benz, the most soluble of the compounds. The Cu2+ sequestering potential of this hydrazone towards Aß was explored by 1H NMR. Although increasing amounts of X1Benz were unexpectedly not efficient at removing the metal-induced perturbations in Aß backbone amides, the broadening effects observed on the compound's signals indicate the formation of a ternary Aß­copper-X1Benz species, which can be responsible for the observed ROS-lessening and aggregation-preventing activities. Overall, the N-acylhydrazones X1TMP and X1Benz have shown promising prospects as agents for the treatment of AD.

Assuntos

Doença de Alzheimer , Humanos , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Cobre/química , Mescalina , Espécies Reativas de Oxigênio/metabolismo , Peptídeos beta-Amiloides/química

RESUMO

Recent studies revealed that molecular events related with the physiology and pathology of αS might be regulated by specific sequence motifs in the primary sequence of αS. The importance of individual residues in these motifs remains an important open avenue of investigation. In this work, we have addressed the structural details related to the amyloid fibril assembly and lipid-binding features of αS through the design of site-directed mutants at position 39 of the protein and their study by in vitro and in vivo assays. We demonstrated that aromaticity at position 39 of αS primary sequence influences strongly the aggregation properties and the membrane-bound conformations of the protein, molecular features that might have important repercussions for the function and dysfunction of αS. Considering that aggregation and membrane damage is an important driver of cellular toxicity in amyloid diseases, future work is needed to link our findings with studies based on toxicity and neuronal cell death. BRIEF STATEMENT OUTLINING SIGNIFICANCE: Modulation by distinct sequential motifs and specific residues of αS on its physiological and pathological states is an active area of research. Here, we demonstrated that aromaticity at position 39 of αS modulates the membrane-bound conformations of the protein, whereas removal of aromatic functionality at position 39 reduces strongly the amyloid assembly in vitro and in vivo. Our study provides new evidence for the modulation of molecular events related with the physiology and pathology of αS.

Assuntos

Amiloide , alfa-Sinucleína , Amiloide/genética , Amiloide/metabolismo , Membranas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , alfa-Sinucleína/química

RESUMO

Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease (PD). Copper ions specifically bind at the N-terminus of AS, accelerating protein aggregation. Its protein homolog ß-synuclein (BS) is also a copper binding protein, but it inhibits AS aggregation. Here, a comparative spectroscopic study of the Cu2+ binding properties of AS and BS has been performed, using electronic absorption, circular dichroism (CD) and electronic paramagnetic resonance (EPR). Our comparative spectroscopic study reveals striking similarities between the Cu2+ binding features of the two proteins. The Cu2+ binding site at the N-terminal group of BS protein, modeled by the BS (1-15) fragment is identical to that of AS; however, its rate of reduction is three times faster as compared to the AS site, consistent with BS having an additional Met residue in its Met1-Xn-Met5-Xn-Met10 motif. The latter is also evident in the cyclic voltammetry studies of the Cu-BS complex. On the other hand, the Cu2+ binding features of the His site in both proteins, as modeled by AS(45-55) and BS(60-70), are identical, indicating that the shift in the His position does not affect its coordination features. Finally, replacement of Glu46 by Ala does not alter Cu2+ binding to the His site, suggesting that the familial PD E46K mutation would not impact copper-induced aggregation. While further studies of the redox activity of copper bound to His50 in AS are required to understand the role of this site in metal-mediated aggregation, our study contributes to a better understanding of the bioinorganic chemistry of PD.

Assuntos

Cobre/metabolismo , alfa-Sinucleína/metabolismo , beta-Sinucleína/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Histidina/química , Histidina/metabolismo , Metionina/química , Metionina/metabolismo , Ligação Proteica , alfa-Sinucleína/química , beta-Sinucleína/química

RESUMO

A phytochemical study was performed on three native plant species from the central-western zone of Argentina: Buddleja cordobensis Grisebach, Baccharis salicina Torr. & A. Gray and Nepeta cataria L. We could obtain verbascoside (1) from B. cordobensis. From N. cataria, we could obtain 1, 5, 9-epi-deoxyloganic acid (2) L. Finally, we could isolate 2-ß-(L-rhamnopyranosyl)-3-angeloyloxy-15-acetyloxy-7,13(14)-E-dien-ent-labdane (3) and 2-ß-(L-rhamnopyranosyl)-3-α-angeloyloxy-15-hydroxy-7,13(14)-E-dien-ent-labdane (4) from B. salicina. Moreover, three derivatives from 1, and one semi-synthetic derivative from 2, were prepared. PCR reaction was used to analyse the activity against DNA polymerase and cell culture to determine cytotoxicity and antitumoral activity. Verbascoside (1) was strongly active in the nanomolar scale (IC50 = 356 nM) against DNA polymerization. Moreover, verbascoside was also strongly active in the nanomolar scale against human melanoma cell line (IC50 = 256 nM) and human colorectal cell line (IC50 = 320 nM). Furthermore, derivatives 6 and 7 were cytotoxic against both cancer cell lines.

Assuntos

Buddleja , Glicosídeos , Glucosídeos/farmacologia , Glicosídeos/farmacologia , Humanos , Fenóis

RESUMO

Although normal aging presents an accumulation of copper and iron in the brain, this becomes more relevant in neurodegeneration. α-Synuclein (α-Syn) misfolding has long been linked with the development of Parkinson's disease (PD). Copper binding promotes aggregation of α-Syn, as well as generalized oxidative stress. In this sense, the use of therapies that target metal dyshomeostasis has been in focus in the past years. Metal-Protein Attenuating Compounds (MPACs) are moderate chelators that aim at disrupting specific, abnormal metal-protein interactions. Our research group has now established that N-acylhydrazones compose a set of truly encouraging MPACs for the bioinorganic management of metal-enhanced aggregopathies. In the present work, a novel ligand, namely 1-methyl-1H-imidazole-2-carboxaldehyde isonicotinoyl hydrazone (X1INH), is reported. We describe solution studies on the interaction and affinity of this compound for copper(ii) ions showing that a fine tuning of metal-affinity was achieved. A series of in vitro biophysical NMR experiments were performed in order to assess the X1INH ability to compete with α-Syn monomers for the binding of both copper(i) and copper(ii) ions, which are central in PD pathology. A preference for copper(i) has been observed. X1INH is less toxic to human neuroglioma (H4) cells in comparison to structure-related compounds. Finally, we show that treatment with X1INH results in a higher number of smaller, less compact inclusions in a well-established model of α-Syn aggregation. Thus, X1INH constitutes a promising MPAC for the treatment of Parkinson's disease.

Assuntos

Cobre/metabolismo , Hidrazonas/química , Hidrazonas/farmacologia , Agregados Proteicos/efeitos dos fármacos , Sinucleinopatias/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Linhagem Celular , Desenho de Fármacos , Humanos , Ligantes , Ligação Proteica/efeitos dos fármacos , Sinucleinopatias/patologia

RESUMO

The discovery of aggregation inhibitors and the elucidation of their mechanism of action are key in the quest to mitigate the toxic consequences of amyloid formation. We have previously characterized the antiamyloidogenic mechanism of action of sodium phtalocyanine tetrasulfonate ([Na4(H2PcTS)]) on α-Synuclein (αS), demonstrating that specific aromatic interactions are fundamental for the inhibition of amyloid assembly. Here we studied the influence that metal preferential affinity and peripheral substituents may have on the activity of tetrapyrrolic compounds on αS aggregation. For the first time, our laboratory has extended the studies in the field of the bioinorganic chemistry and biophysics to cellular biology, using a well-established cell-based model to study αS aggregation. The interaction scenario described in our work revealed that both N- and C-terminal regions of αS represent binding interfaces for the studied compounds, a behavior that is mainly driven by the presence of negatively or positively charged substituents located at the periphery of the macrocycle. Binding modes of the tetrapyrrole ligands to αS are determined by the planarity and hydrophobicity of the aromatic ring system in the tetrapyrrolic molecule and/or the preferential affinity of the metal ion conjugated at the center of the macrocyclic ring. The different capability of phthalocyanines and meso-tetra (N-methyl-4-pyridyl) porphine tetrachloride ([H2PrTPCl4]) to modulate αS aggregation in vitro was reproduced in cell-based models of αS aggregation, demonstrating unequivocally that the modulation exerted by these compounds on amyloid assembly is a direct consequence of their interaction with the target protein.

Assuntos

Proteínas Amiloidogênicas/metabolismo , Indóis/metabolismo , Porfirinas/metabolismo , Multimerização Proteica/efeitos dos fármacos , Zinco/metabolismo , alfa-Sinucleína/metabolismo , Sequência de Aminoácidos , Proteínas Amiloidogênicas/química , Linhagem Celular Tumoral , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Indóis/química , Indóis/toxicidade , Porfirinas/química , Porfirinas/toxicidade , Ligação Proteica , Zinco/química , alfa-Sinucleína/química

RESUMO

Very few inorganic antineoplastic drugs have entered the clinic in the last decades, mainly because of toxicity issues. Because copper is an essential trace element of ubiquitous occurrence, decreased side effects could be expected in comparison with the widely used platinum anticancer compounds. In the present work, two novel hydrazonic binucleating ligands and their µ-hydroxo dicopper(II) complexes were prepared and fully characterized. They differ by the nature of the aromatic group present in their aroylhydrazone moieties: while H3L1 and its complex, 1, possess a thiophene ring, H3L2 and 2 contain the more polar furan heterocycle. X-ray diffraction indicates that both coordination compounds are very similar in structural terms and generate dimeric arrangements in the solid state. Positive-ion electrospray ionization mass spectrometry analyses confirmed that the main species present in a 10% dimethyl sulfoxide (DMSO)/water solution should be [Cu2(HL)(OH)]+ and the DMSO-substituted derivative [Cu2(L)(DMSO)]+. Scattering techniques [dynamic light scattering (DLS) and small-angle X-ray scattering] suggest that the complexes and their free ligands interact with bovine serum albumin (BSA) in a reversible manner. The binding constants to BSA were determined for the complexes through fluorescence spectroscopy. Moreover, to gain insight into the mechanism of action of the compounds, calf thymus DNA binding studies by UV-visible and DLS measurements using plasmid pBR322 DNA were also performed. For the complexes, DLS data seem to point to the occurrence of DNA cleavage to Form III (linear). Both ligands and their dicopper(II) complexes display potent antiproliferative activity in a panel of four cancer cell lines, occasionally even in the submicromolar range, with the complexes being more potent than the free ligands. Our data on cellular models correlate quite well with the DNA interaction experiments. The results presented herein show that aroylhydrazone-derived binucleating ligands, as well as their dinuclear µ-hydroxodicopper(II) complexes, may represent a promising structural starting point for the development of a new generation of highly active potential antitumor agents.

Assuntos

Antineoplásicos/farmacologia , Complexos de Coordenação/farmacologia , Hidrazonas/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/toxicidade , Bovinos , Linhagem Celular Tumoral , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/toxicidade , Cobre/química , DNA/química , Clivagem do DNA/efeitos dos fármacos , Cães , Humanos , Hidrazonas/síntese química , Hidrazonas/química , Hidrazonas/toxicidade , Isomerismo , Ligantes , Células Madin Darby de Rim Canino , Camundongos , Plasmídeos/química , Multimerização Proteica/efeitos dos fármacos , Soroalbumina Bovina/metabolismo

RESUMO

Parkinson's disease is the second most common neurodegenerative disorder worldwide. Neurodegeneration in this pathology is characterized by the loss of dopaminergic neurons in the substantia nigra, coupled with cytoplasmic inclusions known as Lewy bodies containing α-synuclein. The brain is an organ that concentrates metal ions, and there is emerging evidence that a break-down in metal homeostasis may be a critical factor in a variety of neurodegenerative diseases. α-synuclein has emerged as an important metal-binding protein in the brain, whereas these interactions play an important role in its aggregation and might represent a link between protein aggregation, oxidative damage, and neuronal cell loss. Additionally, α-synuclein undergoes several post-translational modifications that regulate its structure and physiological function, and may be linked to the aggregation and/or oligomer formation. This review is focused on the interaction of this protein with physiologically relevant metal ions, highlighting the cases where metal-AS interactions profile as key modulators for its structural, aggregation, and membrane-binding properties. The impact of α-synuclein phosphorylation and N-terminal acetylation in the metal-binding properties of the protein are also discussed, underscoring a potential interplay between PTMs and metal ion binding in regulating α-synuclein physiological functions and its role in pathology. This article is part of the Special Issue "Synuclein".

Assuntos

Metais/metabolismo , Doença de Parkinson/metabolismo , Processamento de Proteína Pós-Traducional , alfa-Sinucleína/metabolismo , Acetilação , Sítios de Ligação , Encéfalo/metabolismo , Cátions Bivalentes/metabolismo , Humanos , Estresse Oxidativo , Oxigênio/metabolismo , Fosforilação , Agregação Patológica de Proteínas , Ligação Proteica , Domínios Proteicos , Relação Estrutura-Atividade , Sumoilação , alfa-Sinucleína/química

RESUMO

With the increasing life expectancy of the world's population, neurodegenerative diseases, such as Alzheimer's disease (AD), will become a much more relevant public health issue. This fact, coupled with the lack of efficacy of the available treatments, has been driving research directed to the development of new drugs for this pathology. Metal-protein attenuating compounds (MPACs) constitute a promising class of agents with potential application on the treatment of neurodegenerative diseases, such as AD. Currently, most MPACs are based on 8-hydroxyquinoline. Recently, our research group has described the hybrid aroylhydrazone containing the 8-hydroxyquinoline group INHHQ as a promising MPAC. By studying the known structure-related ligand HPCIH, which does not contain the phenol moiety, as a simplified chemical model for INHHQ, we aimed to clarify the real impact of the aroylhydrazone group for the MPAC activity of a compound with potential anti-Alzheimer's activity. The present work describes a detailed solution and solid-state study of the coordination of HPCIH with Zn2+ ions, as well as its in vitro binding-ability towards this metal in the presence of the Aß(1-40) peptide. Similar to INHHQ, HPCIH is able to efficiently compete with Aß(1-40) for Zn2+ ions, performing as expected for an MPAC. The similarity between the behaviors of both ligands is remarkable. Taken together, the data presented herein point to aroylhydrazones, such as the compounds HPCIH and the previously published INHHQ, as encouraging MPACs for the treatment of AD.

Assuntos

Hidrazonas/química , Nootrópicos/química , Piridinas/química , Zinco/química , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/metabolismo , Complexos de Coordenação/síntese química , Complexos de Coordenação/química , Complexos de Coordenação/metabolismo , Hidrazonas/síntese química , Hidrazonas/metabolismo , Ligantes , Estrutura Molecular , Nootrópicos/síntese química , Nootrópicos/metabolismo , Fragmentos de Peptídeos/metabolismo , Estudo de Prova de Conceito , Ligação Proteica , Piridinas/síntese química , Piridinas/metabolismo , Zinco/metabolismo

RESUMO

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 ß-peptide (Aß) and the impact of these interactions on the peptide amyloid fibril assembly. Elucidation of the PcTS binding modes to Aß40 revealed the involvement of specific aromatic and hydrophobic interactions in the formation of the Aß40-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 Aß40-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 Aß40 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.

Assuntos

Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Indóis/metabolismo , Indóis/farmacologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Agregados Proteicos/efeitos dos fármacos , Interações Hidrofóbicas e Hidrofílicas , Isoindóis , Ligação Proteica

RESUMO

The cellular prion protein (PrPC) is a copper binding protein that undergoes post-translational modifications, such as endoproteolytic alpha cleavage, which occurs in the vicinity of the His111 Cu binding site. Alpha cleavage processing of PrPC is considered to be neuroprotective since the cleavage site is located in a region that is key to the conversion of PrPC into the infectious scrapie isoform (PrPSc), yielding a membrane bound C1 fragment of PrPC that still contains His111. In this work, we use hPrP(111-115) fragment as a model peptide to evaluate the impact of alpha cleavage processing of PrPC in its ability to coordinate Cu(ii) ions at His111. By using different spectroscopic techniques such as electronic absorption, circular dichroism, nuclear magnetic resonance, and electron paramagnetic resonance, this study demonstrates that Cu(ii) binding to the cleaved His111 site is highly dependent on Cu and proton concentrations. The imidazole group of His111 and its free NH2 terminus emerge as the main anchoring sites for Cu(ii) coordination, yielding very different complexes from those characterized for the intact His111 site in the full protein. Different Cu(ii) coordination modes that could form with the alpha cleaved PrPC under physiological conditions are identified and characterized. Overall, this study contributes to understand how alpha cleavage processing of PrPC impacts its Cu(ii) binding properties at His111. While the functional implications of Cu binding to the cleaved PrPC remain to be discovered, proteolytic processing of PrPC and its Cu binding features appear to be molecular events that might be strongly linked to its cellular function.

RESUMO

Alterations in the levels of copper in brain tissue and formation of α-synuclein (αS)-copper complexes might play a key role in the amyloid aggregation of αS and the onset of Parkinson's disease (PD). Recently, we demonstrated that formation of the high-affinity Cu(I) complex with the N-terminally acetylated form of the protein αS substantially increases and stabilizes local conformations with α-helical secondary structure and restricted motility. In this work, we performed a detailed NMR-based structural characterization of the Cu(I) complexes with the full-length acetylated form of its homologue ß-synuclein (ßS), which is colocalized with αS in vivo and can bind copper ions. Our results show that, similarly to αS, the N-terminal region of ßS constitutes the preferential binding interface for Cu(I) ions, encompassing two independent and noninteractive Cu(I) binding sites. According to these results, ßS binds the metal ion with higher affinity than αS, in a coordination environment that involves the participation of Met-1, Met-5, and Met-10 residues (site 1). Compared to αS, the shift of His from position 50 to 65 in the N-terminal region of ßS does not change the Cu(I) affinity features at that site (site 2). Interestingly, the formation of the high-affinity ßS-Cu(I) complex at site 1 in the N-terminus promotes a short α-helix conformation that is restricted to the 1-5 segment of the AcßS sequence, which differs with the substantial increase in α-helix conformations seen for N-terminally acetylated αS upon Cu(I) complexation. Our NMR data demonstrate conclusively that the differences observed in the conformational transitions triggered by Cu(I) binding to AcαS and AcßS find a correlation at the level of their backbone dynamic properties; added to the potential biological implications of these findings, this fact opens new avenues of investigations into the bioinorganic chemistry of PD.

Assuntos

Complexos de Coordenação/metabolismo , Cobre/metabolismo , Doença de Parkinson/metabolismo , beta-Sinucleína/metabolismo , Acetilação , Sítios de Ligação , Química Bioinorgânica , Complexos de Coordenação/química , Cobre/química , Humanos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , beta-Sinucleína/química

RESUMO

Alzheimer's and Parkinson's diseases share similar amyloidogenic mechanisms, in which metal ions might play an important role. In this last neuropathy, misfolding and aggregation of α-synuclein (α-Syn) are crucial pathological events. A moderate metal-binding compound, namely, 8-hydroxyquinoline-2-carboxaldehyde isonicotinoyl hydrazone (INHHQ), which was previously reported as a potential 'Metal-Protein Attenuating Compound' for Alzheimer's treatment, is well-tolerated by healthy Wistar rats and does not alter their major organ weights, as well as the tissues' reduced glutathione and biometal levels, at a concentration of 200mgkg-1. INHHQ definitively crosses the blood-brain barrier and can be detected in the brain of rats so late as 24h after intraperitoneal administration. After 48h, brain clearance is complete. INHHQ is able to disrupt, in vitro, anomalous copper-α-Syn interactions, through a mechanism probably involving metal ions sequestering. This compound is non-toxic to H4 (human neuroglioma) cells and partially inhibits intracellular α-Syn oligomerization. INHHQ, thus, shows definite potential as a therapeutic agent against Parkinson's as well.

Assuntos

Barreira Hematoencefálica/metabolismo , Quelantes , Hidrazonas , Doença de Parkinson Secundária/tratamento farmacológico , Animais , Quelantes/síntese química , Quelantes/química , Quelantes/farmacocinética , Quelantes/farmacologia , Avaliação Pré-Clínica de Medicamentos , Hidrazonas/síntese química , Hidrazonas/química , Hidrazonas/farmacocinética , Hidrazonas/farmacologia , Masculino , Doença de Parkinson Secundária/metabolismo , Ratos , Ratos Wistar

RESUMO

Synucleinophaties are progressive neurodegenerative disorders with no cure to date. An attractive strategy to tackle this problem is repurposing already tested safe drugs against novel targets. In this way, doxycycline prevents neurodegeneration in Parkinson models by modulating neuroinflammation. However, anti-inflammatory therapy per se is insufficient to account for neuroprotection. Herein we characterise novel targets of doxycycline describing the structural background supporting its effectiveness as a neuroprotector at subantibiotic doses. Our results show that doxycycline reshapes α-synuclein oligomers into off-pathway, high-molecular-weight species that do not evolve into fibrils. Off-pathway species present less hydrophobic surface than on-pathway oligomers and display different ß-sheet structural arrangement. These structural changes affect the α-synuclein ability to destabilize biological membranes, cell viability, and formation of additional toxic species. Altogether, these mechanisms could act synergically giving novel targets for repurposing this drug.

Assuntos

Doxiciclina/farmacologia , Reposicionamento de Medicamentos , Doenças Neurodegenerativas/metabolismo , alfa-Sinucleína/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular , Doxiciclina/uso terapêutico , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/patologia , Agregados Proteicos/efeitos dos fármacos , Agregação Patológica de Proteínas , Ligação Proteica , Conformação Proteica em Folha beta , Multimerização Proteica , Espectroscopia de Infravermelho com Transformada de Fourier , alfa-Sinucleína/química

RESUMO

Human islet amyloid polypeptide (hIAPP) is the major component of amyloid deposits found in pancreatic ß-cells of patients with type 2 diabetes (T2D). Copper ions have an inhibitory effect on the amyloid aggregation of hIAPP, and they may play a role in the etiology of T2D. However, deeper knowledge of the structural details of the copper-hIAPP interaction is required to understand the molecular mechanisms involved. Here, we performed a spectroscopic study of Cu(II) binding to hIAPP and several variants, using electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR), electronic absorption, and circular dichroism (CD) in the UV-vis region in combination with Born-Oppenheimer molecular dynamics (BOMD) and density functional theory geometry optimizations. We find that Cu(II) binds to the imidazole N1 of His18, the deprotonated amides of Ser19 and Ser20, and an oxygen-based ligand provided by Ser20, either via its hydroxyl group or its backbone carbonyl, while Asn22 might also play a role as an axial ligand. Ser20 plays a crucial role in stabilizing Cu(II) coordination toward the C-terminal, providing a potential link between the S20G mutation associated with early onset of T2D, its impact in Cu binding properties, and hIAPP amyloid aggregation. Our study defines the nature of the coordination environment in the Cu(II)-hIAPP complex, revealing that the amino acid residues involved in metal ion binding are also key residues for the formation of ß-sheet structures and amyloid fibrils. Cu(II) binding to hIAPP may lead to the coexistence of more than one coordination mode, which in turn could favor different sets of Cu-induced conformational ensembles. Cu-induced hIAPP conformers would display a higher energetic barrier to form amyloid fibrils, hence explaining the inhibitory effect of Cu ions in hIAPP aggregation. Overall, this study provides further structural insights into the bioinorganic chemistry of T2D.

RESUMO

Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease. The interaction of copper ions with the N-terminal region of AS promotes its amyloid aggregation and metal-catalyzed oxidation has been proposed as a plausible mechanism. The AS(1-6) fragment represents the minimal sequence that models copper coordination to this intrinsically disordered protein. In this study, we evaluated the role of methionine residues Met1 and Met5 in Cu(II) coordination to the AS(1-6) fragment, and in the redox activity of the Cu-AS(1-6) complex. Spectroscopic and electronic structure calculations show that Met1 may play a role as an axial ligand in the Cu(II)-AS(1-6) complex, while Met5 does not participate in metal coordination. Cyclic voltammetry and reactivity studies demonstrate that Met residues play an important role in the reduction and reoxidation processes of this complex. However, Met1 plays a more important role than Met5, as substitution of Met1 by Ile decreases the reduction potential of the Cu-AS(1-6) complex by ~80 mV, causing a significant decrease in its rate of reduction. Reoxidation of the complex by oxygen results in oxidation of the Met residues to sulfoxide, being Met1 more susceptible to copper-catalyzed oxidation than Met5. The sulfoxide species can suffer elimination of methanesulfenic acid, rendering a peptide with no thioether moiety, which would impair the ability of AS to bind Cu(I) ions. Overall, our study underscores the important roles that Met1 plays in copper coordination and the reactivity of the Cu-AS complex.

Assuntos

Cobre/química , Metionina/química , alfa-Sinucleína/química , Humanos , Cinética , Estrutura Molecular

RESUMO

The aggregation of proteins into toxic conformations plays a critical role in the development of different neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Creutzfled-Jakob's disease (CJD). These disorders share a common pathological mechanism that involves the formation of aggregated protein species including toxic oligomers and amyloid fibrils. The aggregation of alpha-synuclein (αS) in PD and the amyloid beta peptide (Aß) and tau protein in AD results in neuronal death and disease onset. In the case of CJD, the misfolding of the physiological prion protein (PrP) induces a chain reaction that results in accumulation of particles that elicit brain damage. Currently, there is no preventive therapy for these diseases and the available therapeutic approaches are based on the treatment of the symptoms rather than the underlying causes of the disease. Accordingly, the aggregation pathway of these proteins represents a useful target for therapeutic intervention. Therefore, understanding the mechanism of amyloid formation and its inhibition is of high clinical importance. The design of small molecules that efficiently inhibit the aggregation process and/or neutralize its associated toxicity constitutes a promising tool for the development of therapeutic strategies against these disorders. In this accounts, we discuss current knowledge on the anti-amyloid activity of phthalocyanines and their potential use as drug candidates in neurodegeneration. These tetrapyrrolic compounds modulate the amyloid assembly of αS, tau, Aß, and the PrP in vitro, and protect cells from the toxic effects of amyloid aggregates. In addition, in scrapie-infected mice, these compounds showed important prophylactic antiscrapie properties. The structural basis for the inhibitory effect of phthalocyanines on amyloid filament assembly relies on specific π-π interactions between the aromatic ring system of these molecules and aromatic residues in the amyloidogenic proteins. Analysis of the structure-activity relationship in phthalocyanines revealed that their anti-amyloid activity is highly dependent on the type of metal ion coordinated to the tetrapyrrolic system but is not sensitive to the number of peripheral charged substituents. The tendency of phthalocyanines to oligomerize (self-association) via aromatic-aromatic stacking interactions correlates precisely with their binding capabilities to target proteins and, more importantly, determines their efficiency as anti-amyloid agents. The ability to block different types of disease-associated protein aggregation raises the possibility that these cyclic tetrapyrrole compounds have a common mechanism of action to impair the formation of a variety of pathological aggregates. Because the structural and molecular basis for the anti-amyloid effects of these molecules is starting to emerge, combined efforts from the fields of structural, cellular, and animal biology will result critical for the rational design and discovery of new drugs for the treatment of amyloid related neurological disorders.

Assuntos

Indóis/química , Doenças Neurodegenerativas/metabolismo , Proteínas/metabolismo , Humanos , Isoindóis , Ligação Proteica , Proteínas/química , Relação Estrutura-Atividade

RESUMO

Amyloid aggregation of α-synuclein (AS) has been linked to the pathological effects associated with Parkinson's disease (PD). Cu(II) binds specifically at the N-terminus of AS and triggers its aggregation. Site-specific Cu(I)-catalyzed oxidation of AS has been proposed as a plausible mechanism for metal-enhanced AS amyloid formation. In this study, Cu(I) binding to AS was probed by NMR spectroscopy, in combination with synthetic peptide models, site-directed mutagenesis, and C-terminal-truncated protein variants. Our results demonstrate that both Met residues in the motif (1)MDVFM(5) constitute key structural determinants for the high-affinity binding of Cu(I) to the N-terminal region of AS. The replacement of one Met residue by Ile causes a dramatic decrease in the binding affinity for Cu(I), whereas the removal of both Met residues results in a complete lack of binding. Moreover, these Met residues can be oxidized rapidly after air exposure of the AS-Cu(I) complex, whereas Met-116 and Met-127 in the C-terminal region remain unaffected. Met-1 displays higher susceptibility to oxidative damage compared to Met-5 because it is directly involved in both Cu(II) and Cu(I) coordination, resulting in closer exposure to the reactive oxygen species that may be generated by the redox cycling of copper. Our findings support a mechanism where the interaction of AS with copper ions leads to site-specific metal-catalyzed oxidation in the protein under physiologically relevant conditions. In light of recent biological findings, these results support a role for AS-copper interactions in neurodegeneration in PD.

Assuntos

Cobre/química , Doença de Parkinson/metabolismo , alfa-Sinucleína/química , Catálise , Cobre/metabolismo , Oxirredução , Ligação Proteica , Espectroscopia de Prótons por Ressonância Magnética , alfa-Sinucleína/metabolismo

RESUMO

α-Synuclein (AS) aggregation is associated to neurodegeneration in Parkinson's disease (PD). At the same time, alterations in metal ion homeostasis may play a pivotal role in the progression of AS amyloid assembly and the onset of PD. Elucidation of the structural basis directing AS-metal interactions and their effect on AS aggregation constitutes a key step towards understanding the role of metal ions in AS amyloid formation and neurodegeneration. Despite of the reported evidences that link Zn(2+) with the pathophysiology of PD and the fact that this metal ion was shown to promote AS fibrillation in vitro, neither the structural characterization of the binding sites nor the identification of the amino acids involved in the interaction of Zn(2+) with the protein AS has been carried out. By using NMR spectroscopy, we have addressed here unknown structural details related to the binding of Zn(2+) to the protein AS through the design of site-directed and domain truncated mutants of AS. The binding of zinc to the Aß peptide was also studied and discussed comparatively. Although the results of this study contribute to the understanding of the structural and molecular basis behind the acceleration of AS fibrillation mediated by Zn(2+), the low affinity that characterizes the interaction of Zn(2+) with AS contrasts strongly with the high-affinity features reported for the binding of this metal ion to other target proteins linked to human amylodosis such as Aß peptide and the Islet Amyloid Polypeptide (IAPP), challenging the biological relevance of zinc interactions in the pathogenesis of PD.

Assuntos

Peptídeos beta-Amiloides/química , Zinco/química , alfa-Sinucleína/química , Sequência de Aminoácidos , Sítios de Ligação , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Relação Estrutura-Atividade , alfa-Sinucleína/genética