RESUMO
We present a novel small molecule antiviral chemotype that was identified by an unconventional cell-free protein synthesis and assembly-based phenotypic screen for modulation of viral capsid assembly. Activity of PAV-431, a representative compound from the series, has been validated against infectious viruses in multiple cell culture models for all six families of viruses causing most respiratory diseases in humans. In animals, this chemotype has been demonstrated efficacious for porcine epidemic diarrhoea virus (a coronavirus) and respiratory syncytial virus (a paramyxovirus). PAV-431 is shown to bind to the protein 14-3-3, a known allosteric modulator. However, it only appears to target the small subset of 14-3-3 which is present in a dynamic multi-protein complex whose components include proteins implicated in viral life cycles and in innate immunity. The composition of this target multi-protein complex appears to be modified upon viral infection and largely restored by PAV-431 treatment. An advanced analog, PAV-104, is shown to be selective for the virally modified target, thereby avoiding host toxicity. Our findings suggest a new paradigm for understanding, and drugging, the host-virus interface, which leads to a new clinical therapeutic strategy for treatment of respiratory viral disease.
Assuntos
Antivirais , Antivirais/farmacologia , Antivirais/química , Humanos , Animais , Proteínas 14-3-3/metabolismo , Complexos Multiproteicos/metabolismo , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Linhagem CelularRESUMO
We present a small molecule chemotype, identified by an orthogonal drug screen, exhibiting nanomolar activity against members of all the six viral families causing most human respiratory viral disease, with a demonstrated barrier to resistance development. Antiviral activity is shown in mammalian cells, including human primary bronchial epithelial cells cultured to an air-liquid interface and infected with SARS-CoV-2. In animals, efficacy of early compounds in the lead series is shown by survival (for a coronavirus) and viral load (for a paramyxovirus). The drug target is shown to include a subset of the protein 14-3-3 within a transient host multi-protein complex containing components implicated in viral lifecycles and in innate immunity. This multi-protein complex is modified upon viral infection and largely restored by drug treatment. Our findings suggest a new clinical therapeutic strategy for early treatment upon upper respiratory viral infection to prevent progression to lower respiratory tract or systemic disease. One Sentence Summary: A host-targeted drug to treat all respiratory viruses without viral resistance development.
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Alzheimer's disease (AD) is a severe neurodegenerative pathology with no effective treatment known. Toxic amyloid-ß peptide (Aß) oligomers play a crucial role in AD pathogenesis. All-d-Enantiomeric peptide D3 and its derivatives were developed to disassemble and destroy cytotoxic Aß aggregates. One of the D3-like compounds is approaching phase II clinical trials; however, high-resolution details of its disease-preventing or pharmacological actions are not completely clear. We demonstrate that peptide D3 stabilizing Aß monomer dynamically interacts with the extracellular juxtamembrane region of a membrane-bound fragment of an amyloid precursor protein containing the Aß sequence. MD simulations based on NMR measurement results suggest that D3 targets the amyloidogenic region, not compromising its α-helicity and preventing intermolecular hydrogen bonding, thus creating prerequisites for inhibition of early steps of Aß conversion into ß-conformation and its toxic oligomerization. An enhanced understanding of the D3 action molecular mechanism facilitates development of effective AD treatment and prevention strategies.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Precursor de Proteína beta-Amiloide/metabolismo , Oligopeptídeos/química , Oligopeptídeos/uso terapêutico , Sequência de Aminoácidos , Animais , Humanos , Camundongos , Simulação de Dinâmica Molecular , Oligopeptídeos/metabolismo , Ligação Proteica , EstereoisomerismoRESUMO
The ability of viruses to evolve several orders of magnitude faster than their host cells has enabled them to exploit host cellular machinery by selectively recruiting multiprotein complexes (MPCs) for their catalyzed assembly and replication. This hijacking may depend on alternative, 'moonlighting' functions of host proteins that deviate from their canonical functions thereby inducing cellular pathology. Here, we posit that if virus-induced cellular pathology is similar to that of other, unknown (non-viral) causes, the identification and molecular characterization of the host proteins involved in virus-mediated cellular pathology can be leveraged to decipher the non-viral disease-relevant mechanisms. We focus on how virus-induced aberrant proteostasis and protein aggregation resemble the cellular pathology of sporadic neurodegenerative diseases (NDs) and how this can be exploited for drug discovery.
Assuntos
Encéfalo , Vírus , Encéfalo/patologia , Encéfalo/virologia , Humanos , Complexos Multiproteicos , Agregação Patológica de Proteínas , ProteostaseRESUMO
OBJECTIVES: The heterogeneity of Amyloid-beta (Aß) plaque load in patients with Alzheimer's disease (AD) has puzzled neuropathology. Since brain Aß plaque load does not correlate with cognitive decline, neurotoxic soluble Aß oligomers have been championed as disease-causing agents in early AD. So far, investigating molecular interactions between soluble oligomeric Aß and insoluble Aß in vivo has been difficult because of the abundance of Aß oligomer species and the kinetic equilibrium in which they coexist. Here, we investigated whether Aß plaque heterogeneity relates to interactions of different Aß conformers. MATERIALS AND METHODS: We took advantage of transgenic mice that generate exclusively Aß dimers (tgDimer mice) but do not develop Aß plaques or neuroinflammation during their lifetime, crossed them to the transgenic CRND8 mice that develop plaques after 90 days and measured Aß plaque load using immunohistochemical and biochemical assays. Furthermore, we performed in vitro thioflavin T (ThT) aggregation assays titrating synthetic Aß42 -S8C dimers into fibril-forming synthetic Aß42 . RESULTS: We observed a lower number of Aß plaques in the brain of double transgenic mice compared to tgCRND8 mice alone while the average plaque size remained unaltered. Corroborating these in vivo findings, synthetic Aß-S8C dimers inhibited fibril formation of wild-type Aß also in vitro, seen by an increased half-time in the ThT assay. CONCLUSIONS: Our study indicates that Aß dimers directly interfere with Aß fibril formation in vivo and in vitro. The variable interaction of Aß dimers with insoluble Aß seeds could thus contribute to the heterogeneity of Aß plaque load in AD patients.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Disfunção Cognitiva/patologia , Humanos , Camundongos Transgênicos , Doenças Neuroinflamatórias/metabolismo , Doenças Neuroinflamatórias/patologia , Fragmentos de Peptídeos/metabolismoRESUMO
COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Düsseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within 2 days of exposure. We identified that SARS-CoV-2 preferably targets neurons of brain organoids. Imaging neurons of organoids reveal that SARS-CoV-2 exposure is associated with altered distribution of Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Our studies, therefore, provide initial insights into the potential neurotoxic effect of SARS-CoV-2 and emphasize that brain organoids could model CNS pathologies of COVID-19.
Assuntos
Betacoronavirus/fisiologia , Encéfalo/virologia , Neurônios/virologia , Animais , Morte Celular , Chlorocebus aethiops , Humanos , Doenças do Sistema Nervoso/virologia , Organoides , SARS-CoV-2 , Células Vero , Proteínas tau/metabolismoRESUMO
Genes associated with immune response and inflammation have been identified as genetic risk factors for late-onset Alzheimer´s disease (LOAD). The rare R47H variant within triggering receptor expressed on myeloid cells 2 (TREM2) has been shown to increase the risk for developing Alzheimer's disease (AD) 2-3-fold. Here, we report the generation and characterization of a model of late-onset Alzheimer's disease (LOAD) using lymphoblast-derived induced pluripotent stem cells (iPSCs) from patients carrying the TREM2 R47H mutation, as well as from control individuals without dementia. All iPSCs efficiently differentiated into mature neuronal cultures, however AD neuronal cultures showed a distinct gene expression profile. Furthermore, manipulation of the iPSC-derived neuronal cultures with an Aß-S8C dimer highlighted metabolic pathways, phagosome and immune response as the most perturbed pathways in AD neuronal cultures. Through the construction of an Aß-induced gene regulatory network, we were able to identify an Aß signature linked to protein processing in the endoplasmic reticulum (ER), which emphasized ER-stress, as a potential causal role in LOAD. Overall, this study has shown that our AD-iPSC based model can be used for in-depth studies to better understand the molecular mechanisms underlying the etiology of LOAD and provides new opportunities for screening of potential therapeutic targets.
Assuntos
Doença de Alzheimer/genética , Glicoproteínas de Membrana/genética , Receptores Imunológicos/genética , Idoso , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Diferenciação Celular/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Feminino , Redes Reguladoras de Genes/genética , Predisposição Genética para Doença/genética , Variação Genética/genética , Humanos , Masculino , Glicoproteínas de Membrana/metabolismo , Modelos Biológicos , Mutação/genética , Células Mieloides/metabolismo , Neurônios/metabolismo , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Receptores Imunológicos/metabolismoRESUMO
Neurodegenerative diseases feature specific misfolded or misassembled proteins associated with neurotoxicity. The precise mechanisms by which protein aggregates first arise in the majority of sporadic cases have remained unclear. Likely, a first critical mass of misfolded proteins starts a vicious cycle of a prion-like expansion. We hypothesize that viruses, having evolved to hijack the host cellular machinery for catalyzing their replication, lead to profound disturbances of cellular proteostasis, resulting in such a critical mass of protein aggregates. Here, we investigated the effect of influenza virus (H1N1) strains on proteostasis of proteins associated with neurodegenerative diseases in Lund human mesencephalic dopaminergic cells in vitro and infection of Rag knockout mice in vivo. We demonstrate that acute H1N1 infection leads to the formation of α-synuclein and Disrupted-in-Schizophrenia 1 (DISC1) aggregates, but not of tau or TDP-43 aggregates, indicating a selective effect on proteostasis. Oseltamivir phosphate, an antiinfluenza drug, prevented H1N1-induced α-synuclein aggregation. As a cell pathobiological mechanism, we identified H1N1-induced blocking of autophagosome formation and inhibition of autophagic flux. In addition, α-synuclein aggregates appeared in infected cell populations connected to the olfactory bulbs following intranasal instillation of H1N1 in Rag knockout mice. We propose that H1N1 virus replication in neuronal cells can induce seeds of aggregated α-synuclein or DISC1 that may be able to initiate further detrimental downstream events and should thus be considered a risk factor in the pathogenesis of synucleinopathies or a subset of mental disorders. More generally, aberrant proteostasis induced by viruses may be an underappreciated factor in initiating protein misfolding.
Assuntos
Proteínas de Homeodomínio/fisiologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/complicações , Infecções por Orthomyxoviridae/complicações , Proteostase , Sinucleinopatias/etiologia , alfa-Sinucleína/química , Animais , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Feminino , Humanos , Influenza Humana/virologia , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/metabolismo , Infecções por Orthomyxoviridae/virologia , Multimerização Proteica , Sinucleinopatias/metabolismo , Sinucleinopatias/patologia , alfa-Sinucleína/metabolismoRESUMO
We examined behaviors and neurotransmitter levels in the tgDimer mouse, a model for early Alzheimer's disease, that expresses exclusively soluble amyloid beta (Aß) dimers and is devoid of Aß plaques, astrogliosis, and neuroinflammation. Seven-month-old mice were subjected to tests of motor activity, attention, anxiety, habituation learning, working memory, and depression-related behaviors. They were impaired in nonselective attention and motor learning and showed anxiety- and despair-related behaviors. In 7- and 12-month-old mice, levels of acetylcholine, dopamine, and serotonin were measured in neostriatum, ventral striatum, prefrontal cortex, hippocampus, amygdala, and entorhinal cortex by high-performance liquid chromatography. The tgDimer mice had lower serotonin turnover rates in hippocampus, ventral striatum, and amygdala relative to wild type controls. The aged tgDimer mice had less hippocampal acetylcholine than adult tgDimers. Stress-test results, based on corticosterone levels, indicated an intact hypothalamus-pituitary-adrenal axis in 12-month-old mice. Since neither Aß plaques nor astrogliosis or neuroinflammation was responsible for these phenotypes, we conclude that Aß dimers contribute to neurotransmitter dysfunction and behavioral impairments, characteristic for the early stages of Alzheimer's disease.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Acetilcolina/metabolismo , Animais , Comportamento Animal , Química Encefálica , Modelos Animais de Doenças , Dopamina/metabolismo , Ácido Hidroxi-Indolacético/metabolismo , Masculino , Camundongos Transgênicos , Serotonina/metabolismo , Estresse Psicológico/metabolismoRESUMO
Accumulating evidence suggests an important role for the Disrupted-in-Schizophrenia 1 (DISC1) protein in neurodevelopment and chronic mental illness. In particular, the C-terminal 300 amino acids of DISC1 have been found to mediate important protein-protein interactions and to harbor functionally important phosphorylation sites and disease-associated polymorphisms. However, long disordered regions and oligomer-forming subdomains have so far impeded structural analysis. VHH domains derived from camelid heavy chain only antibodies are minimal antigen binding modules with appreciable solubility and stability, which makes them well suited for the stabilizing proteins prior to structural investigation. Here, we report on the generation of a VHH domain derived from an immunized Lama glama, displaying high affinity for the human DISC1 C region (aa 691-836), and its characterization by surface plasmon resonance, size exclusion chromatography and immunological techniques. The VHH-DISC1 (C region) complex was also used for structural investigation by small angle X-ray scattering analysis. In combination with molecular modeling, these data support predictions regarding the three-dimensional fold of this DISC1 segment as well as its steric arrangement in complex with our VHH antibody.
Assuntos
Camelídeos Americanos/imunologia , Proteínas do Tecido Nervoso/imunologia , Anticorpos de Cadeia Única/química , Sequência de Aminoácidos , Animais , Complexo Antígeno-Anticorpo/química , Complexo Antígeno-Anticorpo/genética , Reações Antígeno-Anticorpo , Fenômenos Biofísicos , Camelídeos Americanos/genética , Mapeamento de Epitopos , Feminino , Humanos , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Domínios e Motivos de Interação entre Proteínas , Espalhamento a Baixo Ângulo , Anticorpos de Cadeia Única/genética , Ressonância de Plasmônio de Superfície , Difração de Raios XRESUMO
Alzheimer's disease primarily occurs as sporadic disease and is accompanied with vast socio-economic problems. The mandatory basic research relies on robust and reliable disease models to overcome increasing incidence and emerging social challenges. Rodent models are most efficient, versatile, and predominantly used in research. However, only highly artificial and mostly genetically modified models are available. As these 'engineered' models reproduce only isolated features, researchers demand more suitable models of sporadic neurodegenerative diseases. One very promising animal model was the South American rodent Octodon degus, which was repeatedly described as natural 'sporadic Alzheimer's disease model' with 'Alzheimer's disease-like neuropathology'. To unveil advantages over the 'artificial' mouse models, we re-evaluated the age-dependent, neurohistological changes in young and aged Octodon degus (1 to 5-years-old) bred in a wild-type colony in Germany. In our hands, extensive neuropathological analyses of young and aged animals revealed normal age-related cortical changes without obvious signs for extensive degeneration as seen in patients with dementia. Neither significant neuronal loss nor enhanced microglial activation were observed in aged animals. Silver impregnation methods, conventional, and immunohistological stains as well as biochemical fractionations revealed neither amyloid accumulation nor tangle formation. Phosphoepitope-specific antibodies against tau species displayed similar intraneuronal reactivity in both, young and aged Octodon degus.In contrast to previous results, our study suggests that Octodon degus born and bred in captivity do not inevitably develop cortical amyloidosis, tangle formation or neuronal loss as seen in Alzheimer's disease patients or transgenic disease models.
Assuntos
Doença de Alzheimer , Modelos Animais de Doenças , Octodon , Envelhecimento/metabolismo , Envelhecimento/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos Transgênicos , Octodon/metabolismo , Proteínas tau/metabolismoRESUMO
Therapeutically active small molecules represent promising nonimmunogenic alternatives to antibodies for specifically targeting disease-relevant receptors. However, a potential drawback compared to antibody-antigen interactions may be the lower affinity of small molecules toward receptors. Here, we overcome this low-affinity problem by coating the surface of nanoparticles (NPs) with multiple ligands. Specifically, we explored the use of gold and platinum nanoparticles to increase the binding affinity of Aß-specific small molecules to inhibit Aß peptide aggregation into fibrils in vitro. The interactions of bare NPs, free ligands, and NP-bound ligands with Aß are comprehensively studied via physicochemical methods (spectroscopy, microscopy, immunologic tests) and cell assays. Reduction of thioflavin T fluorescence, as an indicator for ß-sheet content, and inhibition of cellular Aß excretion are even more effective with NP-bound ligands than with the free ligands. The results from this study may have implications in the development of therapeutics for treating Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/química , Ligantes , Nanopartículas Metálicas , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/terapia , Ouro , Fragmentos de PeptídeosRESUMO
Bovine spongiform encephalopathy (BSE) created a global European crisis in the 1980s and 90s, with very serious health and economic implications. Classical BSE now appears to be under control, to a great extent as a result of a global research effort that identified the sources of prions in meat and bone meal (MBM) and developed new animal-testing tools that guided policy. Priority ( www.prionpriority.eu ) was a European Union (EU) Framework Program 7 (FP7)-funded project through which 21 European research institutions and small and medium enterprises (SMEs) joined efforts between 2009 and 2014, to conduct coordinated basic and applied research on prions and prion diseases. At the end of the project, the Priority consortium drafted a position paper ( www.prionpriority.eu/Priority position paper) with its main conclusions. In the present opinion paper, we summarize these conclusions. With respect to the issue of re-introducing ruminant protein into the feed-chain, our opinion is that sustaining an absolute ban on feeding ruminant protein to ruminants is essential. In particular, the spread and impact of non-classical forms of scrapie and BSE in ruminants is not fully understood and the risks cannot be estimated. Atypical prion agents will probably continue to represent the dominant form of prion diseases in the near future in Europe. Atypical L-type BSE has clear zoonotic potential, as demonstrated in experimental models. Similarly, there are now data indicating that the atypical scrapie agent can cross various species barriers. More epidemiological data from large cohorts are necessary to reach any conclusion on the impact of its transmissibility on public health. Re-evaluations of safety precautions may become necessary depending on the outcome of these studies. Intensified searching for molecular determinants of the species barrier is recommended, since this barrier is key for important policy areas and risk assessment. Understanding the structural basis for strains and the basis for adaptation of a strain to a new host will require continued fundamental research, also needed to understand mechanisms of prion transmission, replication and how they cause nervous system dysfunction and death. Early detection of prion infection, ideally at a preclinical stage, also remains crucial for development of effective treatment strategies.
Assuntos
Cadeia Alimentar , Doenças Priônicas/epidemiologia , Doenças Priônicas/prevenção & controle , Príons/análise , Ração Animal/efeitos adversos , Animais , Bovinos , Diagnóstico Precoce , Encefalopatia Espongiforme Bovina/diagnóstico , Encefalopatia Espongiforme Bovina/epidemiologia , Encefalopatia Espongiforme Bovina/prevenção & controle , Encefalopatia Espongiforme Bovina/transmissão , Europa (Continente)/epidemiologia , Humanos , Doenças Priônicas/diagnóstico , Doenças Priônicas/transmissão , Príons/isolamento & purificação , Príons/metabolismo , Príons/patogenicidade , Scrapie/diagnóstico , Scrapie/epidemiologia , Scrapie/prevenção & controle , Scrapie/transmissãoRESUMO
Despite amyloid plaques, consisting of insoluble, aggregated amyloid-ß peptides, being a defining feature of Alzheimer's disease, their significance has been challenged due to controversial findings regarding the correlation of cognitive impairment in Alzheimer's disease with plaque load. The amyloid cascade hypothesis defines soluble amyloid-ß oligomers, consisting of multiple amyloid-ß monomers, as precursors of insoluble amyloid-ß plaques. Dissecting the biological effects of single amyloid-ß oligomers, for example of amyloid-ß dimers, an abundant amyloid-ß oligomer associated with clinical progression of Alzheimer's disease, has been difficult due to the inability to control the kinetics of amyloid-ß multimerization. For investigating the biological effects of amyloid-ß dimers, we stabilized amyloid-ß dimers by an intermolecular disulphide bridge via a cysteine mutation in the amyloid-ß peptide (Aß-S8C) of the amyloid precursor protein. This construct was expressed as a recombinant protein in cells and in a novel transgenic mouse, termed tgDimer mouse. This mouse formed constant levels of highly synaptotoxic soluble amyloid-ß dimers, but not monomers, amyloid-ß plaques or insoluble amyloid-ß during its lifespan. Accordingly, neither signs of neuroinflammation, tau hyperphosphorylation or cell death were observed. Nevertheless, these tgDimer mice did exhibit deficits in hippocampal long-term potentiation and age-related impairments in learning and memory, similar to what was observed in classical Alzheimer's disease mouse models. Although the amyloid-ß dimers were unable to initiate the formation of insoluble amyloid-ß aggregates in tgDimer mice, after crossbreeding tgDimer mice with the CRND8 mouse, an amyloid-ß plaque generating mouse model, Aß-S8C dimers were sequestered into amyloid-ß plaques, suggesting that amyloid-ß plaques incorporate neurotoxic amyloid-ß dimers that by themselves are unable to self-assemble. Our results suggest that within the fine interplay between different amyloid-ß species, amyloid-ß dimer neurotoxic signalling, in the absence of amyloid-ß plaque pathology, may be involved in causing early deficits in synaptic plasticity, learning and memory that accompany Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Transtornos Cognitivos/metabolismo , Plasticidade Neuronal/fisiologia , Placa Amiloide/metabolismo , Multimerização Proteica/fisiologia , Peptídeos beta-Amiloides/genética , Animais , Transtornos Cognitivos/genética , Transtornos Cognitivos/patologia , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Cultura de Órgãos , Placa Amiloide/genética , Placa Amiloide/patologiaRESUMO
Determining Aß levels in body fluids remains a powerful tool in the diagnostics of Alzheimer's disease. This report delineates a new supramolecular strategy which increases the affinity of antibodies towards Aß to make diagnostic procedures more sensitive. A monoclonal antibody IC16 was generated to an N-terminal epitope of Aß and the variable regions of the heavy and light chains were cloned as a recombinant protein (scFv). A 6 × histidine tag was fused to the C-terminus of IC16-scFv allowing hybridization with a small organic ß-sheet binder via Ni-NTA complexation. On the other hand, a multivalent nitrilotriacetic acid (NTA)-equipped trimeric aminopyrazole (AP) derivative was synthesized based on a cyclam platform; and experimental evidence was obtained for efficient Ni(2+)-mediated complex formation with the histidine-tagged antibody species. In a proof of principle experiment the hybrid molecule showed a strong increase in affinity towards Aß. Thus, the specific binding power of recombinant antibody fragments to their ß-sheet rich targets can be conveniently enhanced by non-covalent hybridization with small organic ß-sheet binders.
Assuntos
Doença de Alzheimer/diagnóstico , Pirazóis/química , Anticorpos de Cadeia Única/química , Peptídeos beta-Amiloides/química , Catálise , Ensaio de Imunoadsorção Enzimática , Epitopos/química , Histidina/química , Humanos , Hidrogênio/química , Concentração de Íons de Hidrogênio , Fragmentos de Imunoglobulinas/química , Imunoterapia , Ligantes , Espectroscopia de Ressonância Magnética , Níquel/química , Ácido Nitrilotriacético/química , Fragmentos de Peptídeos/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/químicaRESUMO
Viruses can be conceptualized as self-replicating multiprotein assemblies, containing coding nucleic acids. Viruses have evolved to exploit host cellular components including enzymes to ensure their replicative life cycle. New findings indicate that also viral capsid proteins recruit host factors to accelerate their assembly. These assembly machines are RNA-containing multiprotein complexes whose composition is governed by allosteric sites. In the event of viral infection, the assembly machines are recruited to support the virus over the host and are modified to achieve that goal. Stress granules and processing bodies may represent collections of such assembly machines, readily visible by microscopy but biochemically labile and difficult to isolate by fractionation. We hypothesize that the assembly of protein multimers such as encountered in neurodegenerative or other protein conformational diseases, is also catalyzed by assembly machines. In the case of viral infection, the assembly machines have been modified by the virus to meet the virus' need for rapid capsid assembly rather than host homeostasis. In the case of the neurodegenerative diseases, it is the monomers and/or low n oligomers of the so-called aggregated proteins that are substrates of assembly machines. Examples for substrates are amyloid ß peptide (Aß) and tau in Alzheimer's disease, α-synuclein in Parkinson's disease, prions in the prion diseases, Disrupted-in-schizophrenia 1 (DISC1) in subsets of chronic mental illnesses, and others. A likely continuum between virus capsid assembly and cell-to-cell transmissibility of aggregated proteins is remarkable. Protein aggregation diseases may represent dysfunction and dysregulation of these assembly machines analogous to the aberrations induced by viral infection in which cellular homeostasis is pathologically reprogrammed. In this view, as for viral infection, reset of assembly machines to normal homeostasis should be the goal of protein aggregation therapeutics. A key basis for the commonality between viral and neurodegenerative disease aggregation is a broader definition of assembly as more than just simple aggregation, particularly suited for the crowded cytoplasm. The assembly machines are collections of proteins that catalytically accelerate an assembly reaction that would occur spontaneously but too slowly to be relevant in vivo. Being an enzyme complex with a functional allosteric site, appropriated for a non-physiological purpose (e.g. viral infection or conformational disease), these assembly machines present a superior pharmacological target because inhibition of their active site will amplify an effect on their substrate reaction. Here, we present this hypothesis based on recent proof-of-principle studies against Aß assembly relevant in Alzheimer's disease.
Assuntos
Proteínas do Capsídeo/metabolismo , Agregação Patológica de Proteínas/metabolismo , Viroses/metabolismo , Vírus/metabolismo , Animais , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Humanos , Modelos Biológicos , Agregação Patológica de Proteínas/tratamento farmacológico , Montagem de Vírus , Viroses/tratamento farmacológico , Viroses/virologia , Vírus/genéticaRESUMO
BRI2 protein binds amyloid precursor protein to halt amyloid-ß production and inhibits amyloid-ß aggregation via its BRICHOS-domain suggesting a link between BRI2 and Alzheimer's disease (AD). Here, we investigate the possible involvement of BRI2 in human AD pathogenesis. BRI2 containing BRICHOS-domain was increased up to 3-fold in AD hippocampus (p = 0.003, n = 14/group). Immunohistochemistry showed BRI2 deposits associated with amyloid-ß plaques in early pathologic stages (Braak-III; Thal-2/3). We observed a decrease in the protein levels of ADAM10 (p = 0.02) and furin (p = 0.066), as well as an increase in SPPL2b (p < 0.0001) in AD hippocampus. Because these enzymes are involved in BRI2 processing, their changes may lead to aberrant processing of BRI2 promoting its deposition and likely affecting BRI2 function. Loss of BRI2 function in AD was supported by the decreased presence of BRI2-amyloid precursor protein complexes in the hippocampus of AD patients compared with control subjects. In conclusion, our data obtained from human samples indicate that in early stages of AD there is an increased deposition of BRI2, which likely leads to impaired BRI2 function thereby influencing AD pathophysiology.
Assuntos
Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Glicoproteínas de Membrana/fisiologia , Placa Amiloide/genética , Proteínas Adaptadoras de Transdução de Sinal , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Hipocampo/metabolismo , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Ligação Proteica , Estrutura Terciária de ProteínaRESUMO
An emerging common feature of the age-associated neurodegenerative disorders like Alzheimer's disease (AD) and Creutzfeldt-Jakob disease (CJD) is the ability of many disease-associated protein aggregates to induce conversion of a normal counterpart conformer leading to an acceleration of disease progression. Curative pharmacotherapy has not been achieved so far despite successes in elucidating pathomechanisms. Here, we review the pharmaceutical strategy of generating hybrid compounds, i.e. compounds consisting of several independently acting moieties with synergistic effects, on key molecular players in AD and CJD. For prion diseases, we review hybrid compounds consisting of two different heterocyclic compounds, their synergistic effects on prion replication in a cell culture model and their ability to prolong survival of experimentally prion-infected mice in vivo. While a combination therapy of several antiprion compounds including quinacrine, clomipramine, simvastatin and tocopherol prolonged survival time to 10-25%, administration of hybrid compound quinpramine alone, a chimera of acridine and iminodibenzyl scaffolds, led to 10% survival time extension. For AD, we review a hybrid compound consisting of an Aß recognizing D-peptide fused to a small molecule ß-sheet breaker, an aminopyrazole. This molecule was able to diminish Aß oligomers in cell culture and significantly decrease synaptotoxicity as measured by miniature excitatory postsynaptic responses in vitro. Hybrid compounds can dramatically increase potency of their single moieties and lead to novel functions when they act in a simultaneous or sequential manner thereby revealing synergistic properties. Their systematic generation combining different classes of compounds from peptides to small molecules has the potential to significantly accelerate drug discovery.
Assuntos
Compostos Heterocíclicos/farmacologia , Peptídeos/farmacologia , Proteínas PrPSc/antagonistas & inibidores , Doenças Priônicas/tratamento farmacológico , Dobramento de Proteína/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sinergismo Farmacológico , Compostos Heterocíclicos/química , Compostos Heterocíclicos/uso terapêutico , Humanos , Modelos Moleculares , Estrutura Molecular , Peptídeos/química , Peptídeos/uso terapêutico , Proteínas PrPSc/metabolismo , Doenças Priônicas/diagnóstico , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/uso terapêuticoRESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disorder with devastating effects. Currently, therapeutic options are limited to symptomatic treatment. For more than a decade, research focused on immunotherapy for the causal treatment of AD. However, clinical trials with active immunization using Aß encountered severe complications, for example meningoencephalitis. Consequently, attention focused on passive immunization using antibodies. As an alternative to large immunoglobulins (IgGs), Aß binding single-chain variable fragments (scFvs) were used for diagnostic and therapeutic research approaches. scFvs can be expressed in E. coli and may provide improved pharmacokinetic properties like increased blood-brain barrier permeability or reduced side-effects in vivo. In this study, we constructed an scFv from an Aß binding IgG, designated IC16, which binds the N-terminal region of Aß (Aß(1-8)). scFv-IC16 was expressed in E. coli, purified and characterized with respect to its interaction with different Aß species and its influence on Aß fibril formation. We were able to show that scFv-IC16 strongly influenced the aggregation behavior of Aß and could be applied as an Aß detection probe for plaque staining in the brains of transgenic AD model mice. The results indicate potential for therapy and diagnosis of AD.