聚乙二醇对溶菌酶淀粉样纤维形成的作用

目的探索在聚乙二醇(PEG)存在的条件下,溶菌酶淀粉样纤维化及由此生成的聚集体对细胞的毒性作用。方法溶菌酶溶液中分别加入不同相对分子质量的PEG,在pH值为2.0、55℃的条件下孵育。采用硫黄素荧光监测溶菌酶淀粉样纤维化,透射电子显微镜观察聚集体形态,以诱导人红细胞聚集和溶血为指标评估溶菌酶聚集体对细胞膜的损害作用。结果在实验条件下,溶菌酶可形成淀粉样纤维,所有PEG均能够抑制溶菌酶的淀粉样纤维化,改变溶菌酶聚集体的形态,降低聚集体的表面疏水性和聚集体对细胞的损害作用。结论 PEG能够抑制溶菌酶的淀粉样纤维化,并使溶菌酶聚集体的细胞毒性减弱。     

槲皮素对淀粉样纤维细胞毒性的抑制作用

目的采用人红细胞为实验模型,探索槲皮素对溶菌酶淀粉样纤维细胞毒性的抑制作用。方法制备溶菌酶淀粉样纤维,在溶菌酶纤维溶液中加入槲皮素,用原子力显微镜观察槲皮素对淀粉样纤维的分解作用;在红细胞悬液中加入溶菌酶淀粉样纤维和槲皮素,用扫描电镜观察细胞形态;SDS凝胶电泳分离细胞膜蛋白,检测在槲皮素存在的条件下,溶菌酶纤维诱导膜蛋白聚集的作用。结果槲皮素能够破坏淀粉样纤维结构,使纤维解聚,从而使溶菌酶淀粉样纤维的细胞损害作用降低,包括抑制溶菌酶纤维诱导的细胞聚集和细胞膜蛋白交联。结论槲皮素能够破坏成熟的溶菌酶淀粉样纤维结构,抑制淀粉样纤维对细胞膜的损害作用。槲皮素的这种作用与其分子的疏水性和抗氧化作用有关。     

Benzofuranone derivatives as effective small molecules related to insulin amyloid fibrillation: a structure-function study

Amyloids are protein fibrils of nanometer size resulting from protein self-assembly. They have been shown to be associated with a wide variety of diseases such as Alzheimer's and Parkinson's and may contribute to various other pathological conditions, known as amyloidoses. Insulin is prone to form amyloid fibrils under slightly destabilizing conditions in vitro and may form amyloid structures when subcutaneously injected into patients with diabetes. There is a great deal of interest in developing novel small molecule inhibitors of amyloidogenic processes, as potential therapeutic compounds. In this study, the effects of five new synthetic benzofuranone derivatives were investigated on the insulin amyloid formation process. Protein fibrillation was analyzed by thioflavin-T fluorescence, Congo red binding, circular dichroism, and electron microscopy. Despite high structural similarity, one of the five tested compounds was observed to enhance amyloid fibrillation, while the others inhibited the process when used at micromolar concentrations, which could make them interesting potential lead compounds for the design of therapeutic antiamyloidogenic compounds.     

Synthesis and in vitro characterization of some benzothiazole- and benzofuranone-derivatives for quantification of fibrillar aggregates and inhibition of amyloid-mediated peroxidase activity

Neurodegenerative diseases are characterized by amyloid deposition. Thioflavin T (ThT) is one of the molecules considered for detection of amyloid deposits; however, its lipophilicity is too low to cross the blood–brain barrier. Therefore, there is a strong motivation to develop suitable compounds for in vitro fibril quantification as well as for in vivo amyloid imaging. Moreover, oxidative stress (particularly, uncontrolled peroxidase activity) has frequently been reported to play a critical role in the onset/progression of some neurodegenerative disorders. In this study, we describe the synthesis of some benzothiazole and benzofuranone compounds and examine their peroxidase inhibitory properties. Furthermore, to establish the potential binding of synthesized compounds to amyloid aggregates, their in vitro binding to some non-disease related amyloidogenic proteins were characterized. Analyses of the in vitro binding studies indicated that compounds 2 and 4 bind to the amyloid structures successfully while compounds 1 and 3 showed a low affinity in binding to fibrils. Furthermore, compounds 3 and 4 were observed to inhibit amyloid-mediated peroxidase activity in a reversible un-competitive manner.     

谷胱甘肽对胰岛素淀粉样纤维化的抑制作用

目的探索谷胱甘肽抑制胰岛素淀粉样纤维化及纤维细胞毒性的分子机制。方法在pH 2.0、37℃及90r·min-1震荡的条件下孵育胰岛素,采用硫黄素(ThT)荧光检测胰岛素形成淀粉样纤维的动力学曲线,8-苯胺-1-萘磺酸(ANS)荧光检测胰岛素分子聚集体表面疏水性的变化,透射电镜观察纤维形态,以淀粉样纤维诱导人红细胞的聚集为指标,评估谷胱甘肽对胰岛素纤维细胞毒性的抑制作用。结果胰岛素在本文的实验条件下孵育可形成淀粉样纤维,谷胱甘肽能够抑制胰岛素的淀粉样纤维化和降低形成的纤维聚集体的表面疏水性,并降低胰岛素纤维对细胞的损害作用。谷胱甘肽的这种作用与分子中的巯基相关。结论谷胱甘肽能够抑制胰岛素的淀粉样纤维化,改变胰岛素聚集体的表面特性,从而使聚集体的细胞毒性降低。     

A New Series of 1,3‐Dihidro‐Imidazo[1,5‐c]thiazole‐5,7‐Dione Derivatives: Synthesis and Interaction with Aβ(25‐35) Amyloid Peptide

Deposition of senile plaques composed of fibrillar aggregates of Aβ‐amyloid peptide is a characteristic hallmark of Alzheimer’s disease. A widely employed approach in the study of anti‐Alzheimer agents involves the identification of substances able to prevent amyloid aggregation, or to disaggregate the amyloid fibrils through a direct structural interaction with the soluble or aggregated forms of the peptide. Here, we report the synthesis of a set of 1,3‐dihydro‐3,6‐disubstituted‐imidazo[1,5‐c]thiazole‐5,7‐dione derivatives supporting different alkyl, aryl and alkylamine side chains. The ability of these compounds to interact with the Aβ(25‐35) peptide was evaluated using circular dichroism, nuclear magnetic resonance and thioflavin fluorescence spectroscopy. A molecular model for Aβ(25‐35)–ligand interactions was calculated by molecular docking procedures. Our data show that the ability of the synthesized compounds to modify the structural behaviour of Aβ(25‐35) varies as a function of the overall structural features of the ligands rather contributions from specific individual substituents.     

Protein conformational diseases: from mechanisms to drug designs

Amyloidosis comprises a group of diseases characterized by the deposition of insoluble protein fibrils in specific organs and includes several serious medical disorders, such as Alzheimer's disease, prion-associated transmissible spongiform encephalitis, and type II diabetes. Despite the structural dissimilarity between the soluble proteins and peptides, these fibrils exhibit similar morphologies under electron microscopy with a characteristic "cross beta-sheet" pattern examined by x-ray fiber diffraction experiments. Many studies have revealed that each of these diseases is associated to a specific protein that is partially unfolded, misfolded, and aggregated. However, the detailed structures of the causative agents and the toxicity mechanisms are less known. This review summarizes recent studies in the conformational disorders leading to aggregation; including which proteins potentially cause conformational diseases, the aggregation mechanisms of these proteins, and recent researches on the conformational changes using advanced experiments or molecular dynamics simulations. Finally, current drug designs towards these protein conformational diseases are also discussed. It is believed that the advances in basic understanding of the mechanisms of conformational changes as well as biological functions of these proteins will shed light on the development and design of potential interfering compounds against amyloid formation associated with protein conformational diseases.     

Novel furan sulphonic acid derivatives for the treatment of neurodegenerative and immunoinflammatory disorders

Furan sulphonic acid derivatives are claimed to inhibit formation of amyloid β-sheets and/or protect against neuronal cell loss and/or inhibit the release of cytokines, such as IL-1β, IL-6 and TNF-α. Thus, these compounds may be useful for the treatment of neurodegenerative diseases, autoimmune diseases and inflammatory diseases.     

芦丁对β淀粉样肽纤维化及细胞毒性的抑制作用

目的探索芦丁对Aβ25-35肽段淀粉样纤维化及纤维细胞毒性的抑制作用。方法在pH值为7.4、温度37℃孵育Aβ25-35肽,采用硫黄素(thioflavin T,ThT)荧光和透射电子显微镜检测多肽的淀粉样纤维化;以淀粉样纤维处理PC12细胞建立的细胞损伤模型,MTT法检测细胞存活率,以评估芦丁对β淀粉样纤维细胞毒性的抑制作用。结果 Aβ25-35肽段在pH值为7.4、温度37℃条件下,经孵育60h左右形成淀粉样纤维;芦丁抑制Aβ淀粉样纤维的形成,破坏纤维结构,并降低纤维诱导的细胞损害。结论芦丁能够抑制Aβ25-35的淀粉样纤维化和破坏成熟纤维结构,并降低Aβ纤维的细胞毒性。     

Characterization of an antibody scFv that recognizes fibrillar insulin and beta-amyloid using atomic force microscopy

Fibrillar amyloid is the hallmark feature of many protein aggregation diseases, such as Alzheimer's and Parkinson's diseases. A monoclonal single-chain variable fragment (scFv) targeting insulin fibrils was isolated using phage display technology and an atomic force microscopy (AFM) mica substrate. Specific targeting of the scFv to insulin fibrils but not monomers or other small oligomeric forms, under similar conditions, was demonstrated both by enzyme-linked immunosorbent assays and AFM recognition imaging. The scFv also recognizes beta-amyloid fibrils, a hallmark feature of Alzheimer's disease. The results suggest that the isolated scFv possibly targets a shared fibrillar motif-probably the cross-beta-sheet characteristic of amyloid fibrils. The techniques outlined here provide additional tools to further study the process of fibril formation. The scFvs isolated can have potential use as diagnostic or therapeutic reagents for protein aggregation diseases.     

Nordihydroguaiaretic acid does not disaggregate beta-amyloid(1-40) protofibrils but does inhibit growth arising from direct protofibril association

Nordihydroguaiaretic acid (NDGA) was observed by Ono et al. (J Neurochem 87:172-181, 2002) to decrease the fluorescence of thioflavin T associated with freshly extended amyloid beta-protein (Abeta) fibrils. They concluded that NDGA could disaggregate Abeta fibrils into aggregates that were larger than monomers or oligomers and did not bind thioflavin T. Such an effect could be of therapeutic importance in the treatment of Alzheimer's disease. In the current study, we confirmed that NDGA induces a decrease in the fluorescence of thioflavin T associated with Abeta(1-40) fibrils and extended this observation to Abeta(1-40) protofibrils. However, attempts to identify protofibril disaggregation products using dynamic light scattering, electron microscopy, and size exclusion chromatography failed to demonstrate any decrease in aggregate size or concentration or a parallel increase in Abeta monomers or small oligomers when protofibrils were incubated with excess NDGA. We propose instead that the decreases in thioflavin T fluorescence resulted from either displacement or conformational alteration of thioflavin T upon the binding of NDGA to these aggregates. In fact, the same equilibrium fluorescence values were observed regardless of the order in which NDGA, thioflavin T, and Abeta protofibrils were added to the incubation. Although NDGA failed to disaggregate Abeta protofibrils, it did inhibit direct protofibril-protofibril association but did not alter protofibril elongation by monomer addition. These results suggest that NDGA might bind along the lateral surface of Abeta protofibrils. In addition, the binding of NDGA to Abeta protofibrils increased their nonspecific adherence to Superdex 75 resin and diminished their effects on cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.     

A specific mechanism of nonspecific inhibition

Promiscuous small molecules plague screening libraries and hit lists. Previous work has found that several nonspecific compounds form submicrometer aggregates, and it has been suggested that this aggregate species is responsible for the inhibition of many different enzymes. It is not understood how aggregates inhibit their targets. To address this question, biophysical, kinetic, and microscopy methods were used to study the interaction of promiscuous, aggregate-forming inhibitors with model proteins. By use of centrifugation and gel electrophoresis, aggregates and protein were found to directly interact. This is consistent with a subsequent observation from confocal fluorescence microscopy that aggregates concentrate green fluorescent protein. beta-Lactamase mutants with increased or decreased thermodynamic stability relative to wild-type enzyme were equally inhibited by an aggregate-forming compound, suggesting that denaturation by unfolding was not the primary mechanism of interaction. Instead, visualization by electron microscopy revealed that enzyme associates with the surface of inhibitor aggregates. This association could be reversed or prevented by the addition of Triton X-100. These observations suggest that the aggregates formed by promiscuous compounds reversibly sequester enzyme, resulting in apparent inhibition. They also suggest a simple method to identify or reverse the action of aggregate-based inhibitors, which appear to be widespread.     

Phosphorus dendrimers affect Alzheimer's (Aβ1-28) peptide and MAP-Tau protein aggregation

Alzheimer's disease (AD) is characterized by pathological aggregation of β-amyloid peptides and MAP-Tau protein. β-Amyloid (Aβ) is a peptide responsible for extracellular Alzheimer's plaque formation. Intracellular MAP-Tau aggregates appear as a result of hyperphosphorylation of this cytoskeletal protein. Small, oligomeric forms of Aβ are intermediate products that appear before the amyloid plaques are formed. These forms are believed to be most neurotoxic. Dendrimers are highly branched polymers, which may find an application in regulation of amyloid fibril formation. Several biophysical and biochemical methods, like circular dichroism (CD), fluorescence intensity of thioflavin T and thioflavin S, transmission electron microscopy, spectrofluorimetry (measuring quenching of intrinsic peptide fluorescence) and MTT-cytotoxicity assay, were applied to characterize interactions of cationic phosphorus-containing dendrimers of generation 3 and generation 4 (CPDG3, CPDG4) with the fragment of amyloid peptide (Aβ(1-28)) and MAP-Tau protein. We have demonstrated that CPDs are able to affect β-amyloid and MAP-Tau aggregation processes. A neuro-2a cell line (N2a) was used to test cytotoxicity of formed fibrils and intermediate products during the Aβ(1-28) aggregation. It has been shown that CPDs might have a beneficial effect by reducing the system toxicity. Presented results suggest that phosphorus dendrimers may be used in the future as agents regulating the fibrilization processes in Alzheimer's disease.     

Small transthyretin (TTR) ligands as possible therapeutic agents in TTR amyloidoses

In transthyretin (TTR) amyloidosis TTR variants deposit as amyloid fibrils giving origin, in most cases, to peripheral polyneuropathy, cardiomyopathy, carpal tunnel syndrome and/or amyloid deposition in the eye. More than eighty TTR variants are known, most of them being pathogenic. The mechanism of TTR fibril formation is still not completely elucidated. However it is widely accepted that the amino acid substitutions in the TTR variants contribute to a destabilizing effect on the TTR tetramer molecule, which in particular conditions dissociate into non native monomeric intermediates that aggregate and polymerize in amyloid fibrils that further elongate. Since this is a multi-step process there is the possibility to impair TTR amyloid fibril formation at different stages of the process namely by tetramer stabilization, inhibition of fibril formation or fibril disruption. Till now the only efficient therapy available is liver transplant when performed in an early phase of the onset of the disease symptoms. Since this is a very invasive therapy alternatives are desirable. In that sense, several compounds have been proposed to impair amyloid formation or disruption. Based on the proposed mechanism for TTR amyloid fibril formation we discuss the action of some of the proposed TTR stabilizers such as derivatives of some NSAIDs (diflunisal, diclofenac, flufenamic acid, and derivatives) and the action of amyloid disrupters such as 4'-iodo-4'-deoxydoxorubicin (I-DOX) and tetracyclines. Among all these compounds, TTR stabilizers seem to be the most interesting since they would impair very early the process of amyloid formation and could also have a prophylactic effect.     

Pulmonary-Allergy,Dermatological, Gastrointestinal & Arthritis: Peptidyl phenylamides as MMP inhibitors

This patent from Syntex discloses novel compounds that inhibit matrix metalloproteinases (MMPs), particularly stromelysin and matrilysin. Accordingly, the compounds are claimed to have utility in the treatment of diseases characterised by an over-expression of MMPs. The compounds of the invention are peptidyl derivatives distinguished by a phenylamide or substituted phenylamide group at the carboxy terminus (P3′ position). A range of conventional zinc chelating groups are claimed, as well as arylthiomethylphosphinic acids which are unique to Syntex.     

Inhibition of aggregate formation as therapeutic target in protein misfolding diseases: effect of tetracycline and trehalose

Importance of the field: The identification of molecules that inhibit protein deposition or reverse fibril formation could open new strategies for therapeutic intervention in misfolding diseases. Numerous compounds have been shown to inhibit amyloid fibril formation in vitro. Among these compounds, tetracycline and the disaccharide trehalose have been reported to inhibit or reverse amyloid aggregation but their efficiency as potential drugs is controversial.

Areas covered in this review: The results obtained using tetracycline and trehalose, reported in the last 15 years, are described and discussed.

What the reader will gain: The conclusions have important implications for the development of therapeutic agents for protein deposition diseases. If fibrillar proteins contribute to cell degeneration, then the disassembly of fibrils may reverse or slow down disease progression; however, if the action of therapeutic agents produces intermediates of fibrillation and/or products of fibril disaggregation, then their accumulation could be harmful.

Take home message: Care should be taken to ensure that strategies aimed at inhibiting fibril formation do not cause a corresponding increase in the concentration of toxic oligomeric species.     

Perspectives for drug intervention in amyloid diseases

Amyloid fibres are stable, persistent and highly ordered aggregates of mis-folded protein that accumulate in tissues and are a prominent feature of the pathology of a wide range of human diseases. The presumed role of amyloid as a causative factor of tissue damage is based largely on 'guilt by association'. However, growing understanding of the nature of amyloid, its formation by a nucleated growth mechanism from destabilised and partially unfolded precursors and its persistence at sites of deposition has provided the foundation for the development of approaches to inhibit amyloid formation and enable its clearance. In spite of intensive study, our understanding of the detailed structure of amyloid itself remains incomplete although 'crossed-beta' structure is clearly a common constituent. On the other hand detailed structural understanding of transthyretin, beta-secretase and serum amyloid P component is contributing to the design of small molecule compounds to target amyloid. Thyroxin mimetics stabilise the native tetrameric protein structure, beta-secretase inhibitors will limit the production of the amyloidogenic Abeta1-42 polypeptide. Compounds that crosslink serum amyloid P component rapidly deplete the plasma and amyloid-bound pool of this protein. The efficacy of these compounds as drugs to prevent formation or enable removal of amyloid will provide a stringent test of the 'amyloid hypothesis' of disease.     

Pharmaceutical strategies against amyloidosis: old and new drugs in targeting a "protein misfolding disease"

The group of diseases caused by abnormalities of the process of protein folding and unfolding is rapidly growing and includes diseases caused by loss of function as well as diseases caused by gain of function of misfolded proteins. Amyloidoses are caused by gain of function of certain proteins that lose their native structure and self-assemble into toxic insoluble, extracellular fibrils. This process requires the contribution of multiple factors of which only a few are established, namely the conformational modification of the amyloidogenic protein, protein's post-translational modifications and the co-deposition of glycosaminoglicans and of serum amyloid P component. In parallel with the exponential growth of biochemical data regarding the key events of the fibrillogenic process, several reports have shown that small molecules, through the interaction with either the amyloidogenic proteins or with the common constituents, can modify the kinetics of formation of amyloid fibrils or can facilitate amyloid reabsorption. These small molecules can be classified on the basis of their protein target and mechanism of action, according to the following properties. 1) molecules that stabilize the amyloidogenic protein precursor 2) molecules that prevent fibrillogenesis by acting on partially folded intermediates of the folding process as well as on low molecular weight oligomers populating the initial phase of fibril formation 3) molecules that interact with mature amyloid fibrils and weaken their structural stability 4) molecules that displace fundamental co-factors of the amyloid deposits like glycosaminoglycans and serum amyloid P component and favor the dissolution of the fibrillar aggregate.     

Turmeric effect on subcutaneous insulin-induced amyloid mass: an in vivo study

Protein-derived amyloid structures are associated with a wide variety of pathologies, including neurodegenerative diseases and local amyloidoses. Reports exist on the ability of insulin to form local amyloidoses under specific conditions. In vitro-generated fibrils of insulin have been previously shown to produce amyloid-containing masses upon repetitive subcutaneous injection in mouse. The present study aimed at investigating the effect of insulin fibrils injection in rats, as well as the potential of turmeric in attenuating this process. It was found that subcutaneous amyloid-containing masses could form in rats at a faster rate compared with mice. Upon addition of turmeric to the fibrils, previous to injection, formed masses had a significantly reduced size, as well as less ordered cellular structure. In conclusion, the results of this study show the potential of turmeric in attenuation of local amyloidosis. Furthermore, we suggest that this model could be of use in screening antiamyloid compounds.     

Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism

The formation of well-ordered fibrillar protein deposits is common to a large group of amyloid-associated disorders. This group consists of several major human diseases such as Alzheimer's disease, Parkinson's disease, prion diseases, and type II diabetes. Currently, there is no approved therapeutic agent directed towards the formation of fibrillar assemblies, which have been recently shown to have a key role in the cytotoxic nature of amyloidogenic proteins. One important approach in the development of therapeutic agents is the use of small molecules that specifically and efficiently inhibit the aggregation process. Several small polyphenol molecules have been demonstrated to remarkably inhibit the formation of fibrillar assemblies in vitro and their associated cytotoxicity. Yet, the inhibition mechanism was mostly attributed to the antioxidative properties of these polyphenol compounds. Based on several observations demonstrating that polyphenols are capable of inhibiting amyloid fibril formation in vitro, regardless of oxidative conditions, and in view of their structural similarities we suggest an additional mechanism of action. This mechanism is assuming structural constraints and specific aromatic interactions, which direct polyphenol inhibitors to the amyloidogenic core. This proposed mechanism is highly relevant for future de novo inhibitors' design as therapeutic agents for the treatment of amyloid-associated diseases.