RESUMO
The Database of Intrinsically Disordered Proteins (DisProt, URL: https://disprot.org) is the major repository of manually curated annotations of intrinsically disordered proteins and regions from the literature. We report here recent updates of DisProt version 9, including a restyled web interface, refactored Intrinsically Disordered Proteins Ontology (IDPO), improvements in the curation process and significant content growth of around 30%. Higher quality and consistency of annotations is provided by a newly implemented reviewing process and training of curators. The increased curation capacity is fostered by the integration of DisProt with APICURON, a dedicated resource for the proper attribution and recognition of biocuration efforts. Better interoperability is provided through the adoption of the Minimum Information About Disorder (MIADE) standard, an active collaboration with the Gene Ontology (GO) and Evidence and Conclusion Ontology (ECO) consortia and the support of the ELIXIR infrastructure.
Assuntos
Bases de Dados de Proteínas , Proteínas Intrinsicamente Desordenadas/metabolismo , Anotação de Sequência Molecular , Software , Sequência de Aminoácidos , DNA/genética , DNA/metabolismo , Conjuntos de Dados como Assunto , Ontologia Genética , Humanos , Internet , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Ligação Proteica , RNA/genética , RNA/metabolismoRESUMO
Parkinson's disease is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra, as well as the accumulation of intraneuronal proteinaceous inclusions known as Lewy bodies and Lewy neurites. The major protein component of Lewy inclusions is the intrinsically disordered protein α-synuclein (α-Syn), which can adopt diverse amyloid structures. Different conformational strains of α-Syn have been proposed to be related to the onset of distinct synucleinopathies; however, how specific amyloid fibrils cause distinctive pathological traits is not clear. Here, we generated three different α-Syn amyloid conformations at different pH and salt concentrations and analyzed the activity of SynuClean-D (SC-D), a small aromatic molecule, on these strains. We show that incubation of α-Syn with SC-D reduced the formation of aggregates and the seeded polymerization of α-Syn in all cases. Moreover, we found that SC-D exhibited a general fibril disaggregation activity. Finally, we demonstrate that treatment with SC-D also reduced strain-specific intracellular accumulation of phosphorylated α-Syn inclusions. Taken together, we conclude that SC-D may be a promising hit compound to inhibit polymorphic α-Syn aggregation.
Assuntos
Fármacos Neuroprotetores/farmacologia , Doença de Parkinson , Piridinas/farmacologia , alfa-Sinucleína , Amiloide/metabolismo , Humanos , Corpos de Lewy/metabolismo , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Polimerização , Agregação Patológica de Proteínas/tratamento farmacológico , Sinucleinopatias/tratamento farmacológico , alfa-Sinucleína/química , alfa-Sinucleína/metabolismoRESUMO
α-Synuclein (α-Syn) aggregates are implicated in Parkinson's disease (PD), so inhibitors of α-Syn aggregation have been intensively explored. It has been demonstrated that small molecules might be able to reduce α-Syn aggregation in fibrils, thus exerting neuroprotective effects in models of PD. To expand our knowledge about the structural requirements for blocking the recognition process into the oligomeric assembly of α-Syn aggregates, we performed a ligand-based virtual screening procedure using two well-known α-Syn aggregation inhibitors, SynuClean-D and ZPD-2, as query compounds. A collection of thirty-four compounds bearing distinct chemical functionalities and mutual chemical features were studied in a Th-T fluorescence test, thus identifying 5-(2,6-dinitro-4-(trifluoromethyl)benzyl)-1-methyl-1H-tetrazole (named MeSC-04) as a potent α-Syn amyloid formation inhibitor that demonstrated similar behavior when compared to SynuClean-D in the thioflavin-T-monitored kinetic assays, with both molecules reducing the number and size of amyloid fibrils, as evidenced by electron microscopy. Molecular modeling studies suggested the binding mode of MeSC-04 through the identification of putative druggable pockets on α-syn fibrils and a subsequent consensus docking methodology. Overall, this work could furnish new insights in the development of α-Syn amyloid inhibitors from synthetic sources.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Humanos , alfa-Sinucleína/metabolismo , Amiloide/metabolismo , Ligantes , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Proteínas AmiloidogênicasRESUMO
The treatment of Parkinson's disease (PD), the second most common neurodegenerative human disorder, continues to be symptomatic. Development of drugs able to stop or at least slowdown PD progression would benefit several million people worldwide. SynuClean-D is a low molecular weight 2-pyridone-based promising drug candidate that inhibits the aggregation of α-synuclein in human cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD. Improving SynuClean-D pharmacokinetic/pharmacodynamic properties, performing structure/activity studies and testing its efficacy in mammalian models of PD requires the use of gr-amounts of the compound. However, not enough compound is on sale, and no synthetic route has been reported until now, which hampers the molecule progress towards clinical trials. To circumvent those problems, we describe here an efficient and economical route that enables the synthesis of SynuClean-D with good yields as well as the synthesis of SynuClean-D derivatives. Structure-activity comparison of the new compounds with SynuClean-D reveals the functional groups of the molecule that can be disposed of without activity loss and those that are crucial to interfere with α-synuclein aggregation. Several of the derivatives obtained retain the parent's compound excellent in vitro anti-aggregative activity, without compromising its low toxicity. Computational predictions and preliminary testing indicate that the blood brain barrier (BBB) permeability of SynuClean-D is low. Importantly, several of the newly designed and obtained active derivatives are predicted to display good BBB permeability. The synthetic route developed here will facilitate their synthesis for BBB permeability determination and for efficacy testing in mammalian models of PD.
Assuntos
Barreira Hematoencefálica/efeitos dos fármacos , Desenho de Fármacos , Doença de Parkinson/tratamento farmacológico , Piridonas/farmacologia , alfa-Sinucleína/antagonistas & inibidores , Animais , Barreira Hematoencefálica/metabolismo , Caenorhabditis elegans , Relação Dose-Resposta a Droga , Estrutura Molecular , Doença de Parkinson/metabolismo , Agregados Proteicos/efeitos dos fármacos , Piridonas/síntese química , Piridonas/química , Relação Estrutura-Atividade , alfa-Sinucleína/metabolismoRESUMO
Parkinson's disease (PD) is characterized by a progressive loss of dopaminergic neurons, a process that current therapeutic approaches cannot prevent. In PD, the typical pathological hallmark is the accumulation of intracellular protein inclusions, known as Lewy bodies and Lewy neurites, which are mainly composed of α-synuclein. Here, we exploited a high-throughput screening methodology to identify a small molecule (SynuClean-D) able to inhibit α-synuclein aggregation. SynuClean-D significantly reduces the in vitro aggregation of wild-type α-synuclein and the familiar A30P and H50Q variants in a substoichiometric molar ratio. This compound prevents fibril propagation in protein-misfolding cyclic amplification assays and decreases the number of α-synuclein inclusions in human neuroglioma cells. Computational analysis suggests that SynuClean-D can bind to cavities in mature α-synuclein fibrils and, indeed, it displays a strong fibril disaggregation activity. The treatment with SynuClean-D of two PD Caenorhabditis elegans models, expressing α-synuclein either in muscle or in dopaminergic neurons, significantly reduces the toxicity exerted by α-synuclein. SynuClean-D-treated worms show decreased α-synuclein aggregation in muscle and a concomitant motility recovery. More importantly, this compound is able to rescue dopaminergic neurons from α-synuclein-induced degeneration. Overall, SynuClean-D appears to be a promising molecule for therapeutic intervention in Parkinson's disease.
Assuntos
Amiloide/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Neurônios Dopaminérgicos/efeitos dos fármacos , Doença de Parkinson/tratamento farmacológico , Agregação Patológica de Proteínas/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , alfa-Sinucleína/antagonistas & inibidores , Amiloide/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Ensaios de Triagem em Larga Escala , Humanos , Neuroblastoma/tratamento farmacológico , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Células Tumorais Cultivadas , alfa-Sinucleína/metabolismoRESUMO
Parkinson's disease is one of the most common neurodegenerative disorders in elderly age. One of the mechanisms involved in the neurodegeneration appears related to the aggregation of the presynaptic protein alpha synuclein (α-syn) into toxic oligomers and fibrils. To date, no highly effective treatment is currently available; therefore, there is an increasing interest in the search of new therapeutic tools. The modulation of α-syn aggregation represents an emergent and promising disease-modifying strategy for reducing or blocking the neurodegenerative process. Herein, by combining in silico and in vitro screenings we initially identified 3-(cinnamylsulfanyl)-5-(4-pyridinyl)-1,2,4-triazol-4-amine (3) as α-syn aggregation inhibitor that was then considered a promising hit for the further design of a new series of small molecules. Therefore, we rationally designed new hit-derivatives that were synthesised and evaluated by biological assays. Lastly, the binding mode of the newer inhibitors was predicted by docking studies.
Assuntos
Aminas/farmacologia , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/farmacologia , Triazóis/farmacologia , alfa-Sinucleína/antagonistas & inibidores , Aminas/síntese química , Aminas/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Humanos , Estrutura Molecular , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Doença de Parkinson/metabolismo , Agregados Proteicos/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/química , alfa-Sinucleína/metabolismoRESUMO
An increasing number of neurodegenerative diseases are being found to be associated with the abnormal accumulation of aggregated proteins in the brain. In Parkinson's disease, this process involves the aggregation of alpha-synuclein (α-syn) into intraneuronal inclusions. Thus, compounds that inhibit α-syn aggregation represent a promising therapeutic strategy as disease-modifying agents for neurodegeneration. The formation of α-syn amyloid aggregates can be reproduced in vitro by incubation of the recombinant protein. However, the in vitro aggregation of α-syn is exceedingly slow and highly irreproducible, therefore precluding fast high throughput anti-aggregation drug screening. Here, we present a simple and easy-to-implement in-plate method for screening large chemical libraries in the search for α-syn aggregation modulators. It allows us to monitor aggregation kinetics with high reproducibility, while being faster and requiring lower protein amounts than conventional aggregation assays. We illustrate how the approach enables the identification of strong aggregation inhibitors in a library of more than 14,000 compounds.
Assuntos
Ensaios de Triagem em Larga Escala/métodos , Bibliotecas de Moléculas Pequenas/farmacologia , alfa-Sinucleína/antagonistas & inibidores , Bases de Dados de Compostos Químicos , Humanos , Cinética , Doença de Parkinson/metabolismo , Proteínas Recombinantes/farmacologiaRESUMO
Functional amyloid (FAs), particularly the bacterial proteins CsgA and FapC, have many useful properties as biomaterials: high stability, efficient, and controllable formation of a single type of amyloid, easy availability as extracellular material in bacterial biofilm and flexible engineering to introduce new properties. CsgA in particular has already demonstrated its worth in hydrogels for stable gastrointestinal colonization and regenerative tissue engineering, cell-specific drug release, water-purification filters, and different biosensors. It also holds promise as catalytic amyloid; existing weak and unspecific activity can undoubtedly be improved by targeted engineering and benefit from the repetitive display of active sites on a surface. Unfortunately, FapC remains largely unexplored and no application is described so far. Since FapC shares many common features with CsgA, this opens the window to its development as a functional scaffold. The multiple imperfect repeats in CsgA and FapC form a platform to introduce novel properties, e.g., in connecting linkers of variable lengths. While exploitation of this potential is still at an early stage, particularly for FapC, a thorough understanding of their molecular properties will pave the way for multifunctional fibrils which can contribute toward solving many different societal challenges, ranging from CO2 fixation to hydrolysis of plastic nanoparticles.
Assuntos
Amiloide , Materiais Biocompatíveis , Animais , Humanos , Amiloide/química , Amiloide/metabolismo , Materiais Biocompatíveis/química , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Hidrogéis/químicaRESUMO
This chapter describes how to test different amyloid preparations for catalytic properties. We describe how to express, purify, prepare and test two types of pathological amyloid (tau and α-synuclein) and two functional amyloid proteins, namely CsgA from Escherichia coli and FapC from Pseudomonas. We therefore preface the methods section with an introduction to these two examples of functional amyloid and their remarkable structural and kinetic properties and high physical stability, which renders them very attractive for a range of nanotechnological designs, both for structural, medical and catalytic purposes. The simplicity and high surface exposure of the CsgA amyloid is particularly useful for the introduction of new functional properties and we therefore provide a computational protocol to graft active sites from an enzyme of interest into the amyloid structure. We hope that the methods described will inspire other researchers to explore the remarkable opportunities provided by bacterial functional amyloid in biotechnology.
Assuntos
Amiloide , Proteínas de Escherichia coli , Escherichia coli , Engenharia de Proteínas , alfa-Sinucleína , Proteínas tau , Amiloide/química , Amiloide/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , alfa-Sinucleína/química , alfa-Sinucleína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Proteínas tau/metabolismo , Proteínas tau/química , Humanos , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Pseudomonas/metabolismo , Pseudomonas/química , Catálise , Domínio CatalíticoRESUMO
Parkinson's disease, the second most common neurodegenerative disorder worldwide, is characterized by the accumulation of protein deposits in the dopaminergic neurons. These deposits are primarily composed of aggregated forms of α-Synuclein (α-Syn). Despite the extensive research on this disease, only symptomatic treatments are currently available. However, in recent years, several compounds, mainly of an aromatic character, targeting α-Syn self-assembly and amyloid formation have been identified. These compounds, discovered by different approaches, are chemically diverse and exhibit a plethora of mechanisms of action. This work aims to provide a historical overview of the physiopathology and molecular aspects associated with Parkinson's disease and the current trends in small compound development to target α-Syn aggregation. Although these molecules are still under development, they constitute an important step toward discovering effective anti-aggregational therapies for Parkinson's disease.
RESUMO
Parkinson's disease, the second most prevalent neurodegenerative disorder worldwide, is characterized by a progressive loss of dopaminergic neurons in substantia nigra pars compacta, causing motor symptoms. This disorder's main hallmark is the formation of intraneuronal protein inclusions, named Lewy bodies and neurites. The major component of these arrangements is α-synuclein, an intrinsically disordered and soluble protein that, in pathological conditions, can form toxic and cell-to-cell transmissible amyloid structures. Preventing α-synuclein aggregation has attracted significant effort in the search for a disease-modifying therapy for Parkinson's disease. Small molecules like SynuClean-D, epigallocatechin gallate, trodusquemine, or anle138b exemplify this therapeutic potential. Here, we describe a subset of compounds containing a single aromatic ring, like dopamine, ZPDm, gallic acid, or entacapone, which act as molecular chaperones against α-synuclein aggregation. The simplicity of their structures contrasts with the complexity of the aggregation process, yet the block efficiently α-synuclein assembly into amyloid fibrils, in many cases, redirecting the reaction towards the formation of non-toxic off-pathway oligomers. Moreover, some of these compounds can disentangle mature α-synuclein amyloid fibrils. Their simple structures allow structure-activity relationship analysis to elucidate the role of different functional groups in the inhibition of α-synuclein aggregation and fibril dismantling, making them informative lead scaffolds for the rational development of efficient drugs.
RESUMO
Parkinson's disease (PD) is characterized by the death of dopaminergic neurons. The common histopathological hallmark in PD patients is the formation of intracellular proteinaceous accumulations. The main constituent of these inclusions is alpha-synuclein (α-syn), an intrinsically disordered protein that in pathological conditions creates amyloid aggregates that lead to neurotoxicity and neurodegeneration. The main goal of our study was to optimize our previously identified α-syn aggregation inhibitors of 5-(4-pyridinyl)-1,2,4-triazole chemotype in terms of in vivo efficacy. Our efforts resulted in the identification of ethyl 2-((4-amino-5-(pyridin-4-yl)-4H-1,2,4-triazol-3-yl)thio)acetate (15), which displayed the ability to prevent 1-methyl-4-phenyl-1,2,3,6-tetrahydropiridine-induced bradykinesia as well as to affect the levels of PD markers after the administration of the same neurotoxin. In addition to the in vivo evaluation, for the 5-(4-pyridinyl)-1,2,4-triazole-based compounds, we measured the prevention of the fibrillization process using light scattering and a ThT binding assay; these compounds have been shown to slightly reduce the α-syn aggregation.
Assuntos
Fármacos Neuroprotetores , Doença de Parkinson , Neurônios Dopaminérgicos/metabolismo , Humanos , Fármacos Neuroprotetores/química , Doença de Parkinson/metabolismo , Triazóis/metabolismo , Triazóis/farmacologia , alfa-Sinucleína/metabolismoRESUMO
α-Synuclein aggregation is a key driver of neurodegeneration in Parkinson's disease and related syndromes. Accordingly, obtaining a molecule that targets α-synuclein toxic assemblies with high affinity is a long-pursued objective. Here, we exploit the biophysical properties of toxic oligomers and amyloid fibrils to identify a family of α-helical peptides that bind to these α-synuclein species with low nanomolar affinity, without interfering with the monomeric functional protein. This activity is translated into a high anti-aggregation potency and the ability to abrogate oligomer-induced cell damage. Using a structure-guided search we identify a human peptide expressed in the brain and the gastrointestinal tract with analogous binding, anti-aggregation, and detoxifying properties. The chemical entities we describe here may represent a therapeutic avenue for the synucleinopathies and are promising tools to assist diagnosis by discriminating between native and toxic α-synuclein species.
Assuntos
Amiloide/metabolismo , Doença de Parkinson/patologia , Agregação Patológica de Proteínas/patologia , alfa-Sinucleína/metabolismo , Encéfalo/metabolismo , Trato Gastrointestinal/metabolismo , HumanosRESUMO
Parkinson's disease (PD) is characterized by progressive loss of dopaminergic neurons and the accumulation of deposits of α-synuclein (α-syn) in the brain. The pivotal role of α-syn aggregation in PD makes it an attractive target for potential disease-modifying therapies. However, the disordered nature of the protein, its multistep aggregation mechanism, and the lack of structural information on intermediate species complicate the discovery of modulators of α-syn amyloid deposition. Despite these difficulties, small molecules have been shown to block the misfolding and aggregation of α-syn, and can even disentangle mature α-syn amyloid fibrils. In this review we provide an updated overview of these leading small compounds and discuss how these chemical chaperones hold great promise to alter the course of PD progression.
Assuntos
Amiloide/efeitos dos fármacos , Chaperonas Moleculares/uso terapêutico , Doença de Parkinson/tratamento farmacológico , Preparações Farmacêuticas/administração & dosagem , Bibliotecas de Moléculas Pequenas/uso terapêutico , alfa-Sinucleína/antagonistas & inibidores , HumanosRESUMO
Synucleinopathies are a group of disorders characterized by the accumulation of α-Synuclein amyloid inclusions in the brain. Preventing α-Synuclein aggregation is challenging because of the disordered nature of the protein and the stochastic nature of fibrillogenesis, but, at the same time, it is a promising approach for therapeutic intervention in these pathologies. A high-throughput screening initiative allowed us to discover ZPDm, the smallest active molecule in a library of more than 14.000 compounds. Although the ZPDm structure is highly related to that of the previously described ZPD-2 aggregation inhibitor, we show here that their mechanisms of action are entirely different. ZPDm inhibits the aggregation of wild-type, A30P, and H50Q α-Synuclein variants in vitro and interferes with α-Synuclein seeded aggregation in protein misfolding cyclic amplification assays. However, ZPDm distinctive feature is its strong potency to dismantle preformed α-Synuclein amyloid fibrils. Studies in a Caenorhabditis elegans model of Parkinson's Disease, prove that these in vitro properties are translated into a significant reduction in the accumulation of α-Synuclein inclusions in ZPDm treated animals. Together with previous data, the present work illustrates how different chemical groups on top of a common molecular scaffold can result in divergent but complementary anti-amyloid activities.
RESUMO
α-Synuclein (α-Syn) forms toxic intracellular protein inclusions and transmissible amyloid structures in Parkinson's disease (PD). Preventing α-Syn self-assembly has become one of the most promising approaches in the search for disease-modifying treatments for this neurodegenerative disorder. Here, we describe the capacity of a small molecule (ZPD-2), identified after a high-throughput screening, to inhibit α-Syn aggregation. ZPD-2 inhibits the aggregation of wild-type α-Syn and the A30P and H50Q familial variants in vitro at substoichiometric compound:protein ratios. In addition, the molecule prevents the spreading of α-Syn seeds in protein misfolding cyclic amplification assays. ZPD-2 is active against different α-Syn strains and blocks their seeded polymerization. Treating with ZPD-2 two different PD Caenorhabditis elegans models that express α-Syn either in muscle or in dopaminergic (DA) neurons substantially reduces the number of α-Syn inclusions and decreases synuclein-induced DA neurons degeneration. Overall, ZPD-2 is a hit compound worth to be explored in order to develop lead molecules for therapeutic intervention in PD.
RESUMO
Protein aggregation is responsible for the onset and spread of many human diseases, ranging from neurodegenerative disorders to cancer and diabetes. Moreover, it is one of the major bottlenecks for the production of protein-based therapeutics such as antibodies or enzymes. AGGRESCAN3D (A3D) is a web server aimed to identify and evaluate structural aggregation prone regions, overcoming the limitations of sequence-based algorithms in the prediction of the aggregation propensity of globular proteins. A3D allows the redesign of protein solubility by predicting in silico the impact of mutations and protein conformational fluctuations on the aggregation of native polypeptides.