RESUMO
The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.
Assuntos
Aminoaciltransferases/química , Periodontite/microbiologia , Porphyromonas gingivalis/enzimologia , Prevotella intermedia/enzimologia , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/genética , Aminoaciltransferases/ultraestrutura , Domínio Catalítico/efeitos dos fármacos , Cristalografia por Raios X , Humanos , Periodontite/tratamento farmacológico , Periodontite/genética , Porphyromonas gingivalis/patogenicidade , Prevotella intermedia/patogenicidade , Estrutura Terciária de Proteína/efeitos dos fármacos , Ácido Pirrolidonocarboxílico/química , Ácido Pirrolidonocarboxílico/metabolismo , Tannerella forsythia/enzimologia , Tannerella forsythia/patogenicidadeRESUMO
The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer Disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded ß-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases.
RESUMO
Parkinson's disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by degeneration of dopaminergic neurons of the substantia nigra (SN) and formation of Lewy bodies and Lewy neurites composed of aggregated α-synuclein. Proteolysis of α-synuclein by matrix metalloproteinases was shown to facilitate its aggregation and to affect cell viability. One of the proteolysed fragments, Gln79-α-synuclein, possesses a glutamine residue at its N-terminus. We argue that glutaminyl cyclase (QC) may catalyze the pyroglutamate (pGlu)79-α-synuclein formation and, thereby, contribute to enhanced aggregation and compromised degradation of α-synuclein in human synucleinopathies. Here, the kinetic characteristics of Gln79-α-synuclein conversion into the pGlu-form by QC are shown using enzymatic assays and mass spectrometry. Thioflavin T assays and electron microscopy demonstrated a decreased potential of pGlu79-α-synuclein to form fibrils. However, size exclusion chromatography and cell viability assays revealed an increased propensity of pGlu79-α-synuclein to form oligomeric aggregates with high neurotoxicity. In brains of wild-type mice, QC and α-synuclein were co-expressed by dopaminergic SN neurons. Using a specific antibody against the pGlu-modified neo-epitope of α-synuclein, pGlu79-α-synuclein aggregates were detected in association with QC in brains of two transgenic mouse lines with human α-synuclein overexpression. In human brain samples of PD and dementia with Lewy body subjects, pGlu79-α-synuclein was shown to be present in SN neurons, in a number of Lewy bodies and in dystrophic neurites. Importantly, there was a spatial co-occurrence of pGlu79-α-synuclein with the enzyme QC in the human SN complex and a defined association of QC with neuropathological structures. We conclude that QC catalyzes the formation of oligomer-prone pGlu79-α-synuclein in human synucleinopathies, which may-in analogy to pGlu-Aß peptides in Alzheimer's disease-act as a seed for pathogenic protein aggregation.
Assuntos
Aminoaciltransferases/metabolismo , Sinucleinopatias/genética , alfa-Sinucleína/metabolismo , Animais , Encéfalo/patologia , Sobrevivência Celular , Cromatografia em Gel , Neurônios Dopaminérgicos/metabolismo , Glutamina/metabolismo , Humanos , Cinética , Doença por Corpos de Lewy/genética , Doença por Corpos de Lewy/metabolismo , Camundongos , Camundongos Transgênicos , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Processamento de Proteína Pós-Traducional , Sambucus nigra/citologia , Sambucus nigra/metabolismoRESUMO
The astacin protease Meprin ß represents an emerging target for drug development due to its potential involvement in disorders such as acute and chronic kidney injury and fibrosis. Here, we elaborate on the structural basis of inhibition by a specific Meprin ß inhibitor. Our analysis of the crystal structure suggests different binding modes of the inhibitor to the active site. This flexibility is caused, at least in part, by movement of the C-terminal region of the protease domain (CTD). The CTD movement narrows the active site cleft upon inhibitor binding. Compared with other astacin proteases, among these the highly homologous isoenzyme Meprin α, differences in the subsites account for the unique selectivity of the inhibitor. Although the inhibitor shows substantial flexibility in orientation within the active site, the structural data as well as binding analyses, including molecular dynamics simulations, support a contribution of electrostatic interactions, presumably by arginine residues, to binding and specificity. Collectively, the results presented here and previously support an induced fit and substantial movement of the CTD upon ligand binding and, possibly, during catalysis. To the best of our knowledge, we here present the first structure of a Meprin ß holoenzyme containing a zinc ion and a specific inhibitor bound to the active site. The structural data will guide rational drug design and the discovery of highly potent Meprin inhibitors.
Assuntos
Ácidos Hidroxâmicos/química , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/química , Simulação de Dinâmica Molecular , Inibidores de Proteases/química , Humanos , Relação Estrutura-AtividadeRESUMO
Compelling evidence suggests that pyroglutamate-modified Aß (pGlu3-Aß; AßN3pG) peptides play a pivotal role in the development and progression of Alzheimer's disease (AD). Approaches targeting pGlu3-Aß by glutaminyl cyclase (QC) inhibition (Varoglutamstat) or monoclonal antibodies (Donanemab) are currently in clinical development. Here, we aimed at an assessment of combination therapy of Varoglutamstat (PQ912) and a pGlu3-Aß-specific antibody (m6) in transgenic mice. Whereas the single treatments at subtherapeutic doses show moderate (16-41%) but statistically insignificant reduction of Aß42 and pGlu-Aß42 in mice brain, the combination of both treatments resulted in significant reductions of Aß by 45-65%. Evaluation of these data using the Bliss independence model revealed a combination index of ≈1, which is indicative for an additive effect of the compounds. The data are interpreted in terms of different pathways, in which the two drugs act. While PQ912 prevents the formation of pGlu3-Aß in different compartments, the antibody is able to clear existing pGlu3-Aß deposits. The results suggest that combination of the small molecule Varoglutamstat and a pE3Aß-directed monoclonal antibody may allow a reduction of the individual compound doses while maintaining the therapeutic effect.
Assuntos
Doença de Alzheimer , Aminoaciltransferases/antagonistas & inibidores , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais Murinos/farmacologia , Benzimidazóis/farmacologia , Imidazolinas/farmacologia , Fragmentos de Peptídeos/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Animais , Humanos , Camundongos , Camundongos Transgênicos , Fragmentos de Peptídeos/genéticaRESUMO
Amyloidogenic plaques are hallmarks of Alzheimer's disease (AD) and typically consist of high percentages of modified Aß peptides bearing N-terminally cyclized glutamate residues. The human zinc(II) enzyme glutaminyl cyclase (QC) was shown in vivo to catalyze the cyclization of N-terminal glutamates of Aß peptides in a pathophysiological side reaction establishing QC as a druggable target for therapeutic treatment of AD. Here, we report crystallographic snapshots of human QC catalysis acting on the neurohormone neurotensin that delineate the stereochemical course of catalysis and suggest that hydrazides could mimic the transition state of peptide cyclization and deamidation. This hypothesis is validated by a sparse-matrix inhibitor screening campaign that identifies hydrazides as the most potent metal-binding group compared to classic Zn binders. The structural basis of hydrazide inhibition is illuminated by X-ray structure analysis of human QC in complex with a hydrazide-bearing peptide inhibitor and reveals a pentacoordinated Zn complex. Our findings inform novel strategies in the design of potent and highly selective QC inhibitors by employing hydrazides as the metal-binding warhead.
Assuntos
Doença de Alzheimer/enzimologia , Aminoaciltransferases/antagonistas & inibidores , Aminoaciltransferases/metabolismo , Inibidores Enzimáticos/química , Hidrazinas/química , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Aminoaciltransferases/química , Cristalografia por Raios X , Ciclização/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Hidrazinas/farmacologia , Modelos Moleculares , Terapia de Alvo Molecular , Neurotensina/metabolismo , Conformação Proteica/efeitos dos fármacosRESUMO
Alzheimer's disease (AD) and Parkinson's disease (PD), including dementia with Lewy bodies (DLB), account for the majority of dementia cases worldwide. Interestingly, a significant number of patients have clinical and neuropathological features of both AD and PD, i.e., the presence of amyloid deposits and Lewy bodies in the neocortex. The identification of α-synuclein peptides in amyloid plaques in DLB brain led to the hypothesis that both peptides mutually interact with each other to facilitate neurodegeneration. In this article, we report the influence of Aß(1-42) and pGlu-Aß(3-42) on the aggregation of α-synuclein in vitro. The aggregation of human recombinant α-synuclein was investigated using thioflavin-T fluorescence assay. Fibrils were investigated by means of antibody conjugated immunogold followed by transmission electron microscopy (TEM). Our data demonstrate a significantly increased aggregation propensity of α-synuclein in the presence of minor concentrations of Aß(1-42) and pGlu-Aß(3-42) for the first time, but without effect on toxicity on mouse primary neurons. The analysis of the composition of the fibrils by TEM combined with immunogold labeling of the peptides revealed an interaction of α-synuclein and Aß in vitro, leading to an accelerated fibril formation. The analysis of kinetic data suggests that significantly enhanced nucleus formation accounts for this effect. Additionally, co-occurrence of α-synuclein and Aß and pGlu-Aß, respectively, under pathological conditions was confirmed in vivo by double immunofluorescent labelings in brains of aged transgenic mice with amyloid pathology. These observations imply a cross-talk of the amyloid peptides α-synuclein and Aß species in neurodegeneration. Such effects might be responsible for the co-occurrence of Lewy bodies and plaques in many dementia cases.
Assuntos
Peptídeos beta-Amiloides/química , Agregados Proteicos , alfa-Sinucleína/química , Doença de Alzheimer , Amiloide/química , Amiloide/ultraestrutura , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Animais , Sobrevivência Celular , Imunofluorescência , Cinética , Corpos de Lewy , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Agregação Patológica de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Oligomeric amyloid-ß (Aß) 1-42 disrupts synaptic function at an early stage of Alzheimer's disease (AD). Multiple posttranslational modifications of Aß have been identified, among which N-terminally truncated forms are the most abundant. It is not clear, however, whether modified species can induce synaptic dysfunction on their own and how altered biochemical properties can contribute to the synaptotoxic mechanisms. Here, we show that a prominent isoform, pyroglutamated Aß3(pE)-42, induces synaptic dysfunction to a similar extent like Aß1-42 but by clearly different mechanisms. In contrast to Aß1-42, Aß3(pE)-42 does not directly associate with synaptic membranes or the prion protein but is instead taken up by astrocytes and potently induces glial release of the proinflammatory cytokine TNFα. Moreover, Aß3(pE)-42-induced synaptic dysfunction is not related to NMDAR signalling and Aß3(pE)-42-induced impairment of synaptic plasticity cannot be rescued by D1-agonists. Collectively, the data point to a scenario where neuroinflammatory processes together with direct synaptotoxic effects are caused by posttranslational modification of soluble oligomeric Aß and contribute synergistically to the onset of synaptic dysfunction in AD.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional , Sinapses/fisiologia , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/genética , Animais , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neuroimunomodulação , Plasticidade Neuronal , Fragmentos de Peptídeos/genética , Isoformas de Proteínas , Receptores de N-Metil-D-Aspartato/metabolismo , Transdução de Sinais , Sinapses/metabolismo , Fator de Necrose Tumoral alfa/biossínteseRESUMO
QUALITY ISSUE: Quality assessment is challenging in children with developmental disorders. Previously, a set of quality indicators (QIs) was developed and analyzed in terms of feasibility of use with patients with attention-deficit/hyperactivity disorder (ADHD). QI assessment turned out to be possible but highly complex. Thus, we compared different technologies for automated extraction of data for assessment of QIs. CHOICE OF SOLUTION: Four automated extraction technologies (regular expressions, Apache Solr, Apache Mahout, Apache OpenNLP) were compared with respect to their properties regarding the complexity of implementing the QI, the complexity of implementing a check module, the reliability and quality of results, the complexity of preparation of interdisciplinary medical reports, and the complexity of deployment and installation. IMPLEMENTATION: Twenty medical reports from different institutions were reviewed for compliance with three QIs by these technologies and compared with expert opinions. EVALUATION: Among the four technologies, Apache Solr had the best overall performance. For manual extraction of the three QIs, at least 77 s were necessary per medical report, whereas the prototype evaluated and extracted the QIs automatically in 8 s on average. Unexpectedly, different assessments of the degree of compliance by the experts turned out to be one of the stumbling blocks. An in-depth evaluation compared results on a semantic level. LESSONS LEARNED: It is possible to extract QIs by post-processing automated technologies. This approach can also be applied to other developmental disorders. However, a more uniform documentation throughout institutions involved will be necessary in order to implement this method in daily practice.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Prontuários Médicos , Indicadores de Qualidade em Assistência à Saúde , Criança , Coleta de Dados , Estudos de Viabilidade , HumanosRESUMO
Alzheimer disease is associated with deposition of the amyloidogenic peptide Aß in the brain. Passive immunization using Aß-specific antibodies has been demonstrated to reduce amyloid deposition both in vitro and in vivo Because N-terminally truncated pyroglutamate (pE)-modified Aß species (AßpE3) exhibit enhanced aggregation potential and propensity to form toxic oligomers, they represent particularly attractive targets for antibody therapy. Here we present three separate monoclonal antibodies that specifically recognize AßpE3 with affinities of 1-10 nm and inhibit AßpE3 fibril formation in vitro. In vivo application of one of these resulted in improved memory in AßpE3 oligomer-treated mice. Crystal structures of Fab-AßpE3 complexes revealed two distinct binding modes for the peptide. Juxtaposition of pyroglutamate pE3 and the F4 side chain (the "pEF head") confers a pronounced bulky hydrophobic nature to the AßpE3 N terminus that might explain the enhanced aggregation properties of the modified peptide. The deep burial of the pEF head by two of the antibodies explains their high target specificity and low cross-reactivity, making them promising candidates for the development of clinical antibodies.
Assuntos
Doença de Alzheimer/imunologia , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/imunologia , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Imunoterapia , Ácido Pirrolidonocarboxílico/imunologia , Peptídeos beta-Amiloides/química , Animais , Células Cultivadas , CamundongosRESUMO
Common assays for endoprotease activity of meprin α and ß are based on cleavage of internally quenched substrates. Although direct and convenient, for meprins these assays bear disadvantages such as, e.g., significant substrate inhibition or potential fluorescence quenching by compounds applied in inhibitor analysis. Here, we present a novel continuous assay by introducing an auxiliary enzyme, prolyl tripeptidyl aminopeptidase (PtP) and the chromogenic substrate KKGYVADAP-p-nitroanilide. We provide a quick strategy for expression and one-step-purification of the auxiliary enzyme. The enzyme kinetic data for meprin α and ß suggest hyperbolic v/S-characteristics, the kinetic parameters of substrate conversion by meprin ß were Kmâ¯=â¯184⯱â¯32⯵M and kcatâ¯=â¯20⯱â¯4 s-1. We also present conditions for the use of the fluorogenic substrate KKGYVADAP-AMC to assess meprin ß activity. The assays were applied for determination of inhibitory parameters of the natural inhibitor actinonin and two recently published hydroxamates. Hence, we present two novel methods, which can be applied to assess inhibitory mechanism and potency with the attractive current drug targets meprin α and ß. Furthermore, the assay might also provide implications for analysis of other endoproteases as well as their inhibitors.
Assuntos
Proteínas de Bactérias/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Metaloendopeptidases/análise , Porphyromonas gingivalis/enzimologia , Serina Endopeptidases/metabolismo , Proteínas de Bactérias/química , Dipeptidil Peptidases e Tripeptidil Peptidases/química , Relação Dose-Resposta a Droga , Ácidos Hidroxâmicos/farmacologia , Cinética , Metaloendopeptidases/antagonistas & inibidores , Metaloendopeptidases/metabolismo , Estrutura Molecular , Inibidores de Proteases/farmacologia , Serina Endopeptidases/química , Relação Estrutura-AtividadeRESUMO
Extracellular plaques of amyloid-ß and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer's disease. Plaques comprise amyloid-ß fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer's disease. Despite the importance of plaques to Alzheimer's disease, oligomers are considered to be the principal toxic forms of amyloid-ß. Interestingly, many adverse responses to amyloid-ß, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-ß are strongly associated with Alzheimer's disease, are more toxic than amyloid-ß, residues 1-42 (Aß(1-42)) and Aß(1-40), and have been proposed as initiators of Alzheimer's disease pathogenesis. Here we report a mechanism by which pE-Aß may trigger Alzheimer's disease. Aß(3(pE)-42) co-oligomerizes with excess Aß(1-42) to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aß(1-42) alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aß(3(pE)-42) plus 95% Aß(1-42) (5% pE-Aß) seed new cytotoxic LNOs through multiple serial dilutions into Aß(1-42) monomers in the absence of additional Aß(3(pE)-42). LNOs isolated from human Alzheimer's disease brain contained Aß(3(pE)-42), and enhanced Aß(3(pE)-42) formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aß(3(pE)-42) confers tau-dependent neuronal death and causes template-induced misfolding of Aß(1-42) into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aß(3(pE)-42) acts similarly at a primary step in Alzheimer's disease pathogenesis.
Assuntos
Peptídeos beta-Amiloides/química , Amiloide/toxicidade , Ácido Glutâmico/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/toxicidade , Fragmentos de Peptídeos/química , Príons/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/metabolismo , Amiloide/química , Amiloide/efeitos dos fármacos , Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Animais , Modelos Animais de Doenças , Ácido Glutâmico/química , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Príons/química , Príons/toxicidade , Proteínas tau/deficiência , Proteínas tau/genéticaRESUMO
Passive immunotherapy has emerged as a very promising approach for the treatment of Alzheimer's disease and other neurodegenerative disorders, which are characterized by the misfolding and deposition of amyloid peptides. On the basis of the amyloid hypothesis, the majority of antibodies in clinical development are directed against amyloid ß (Aß), the primary amyloid component in extracellular plaques. This review focuses on the current status of Aß antibodies in clinical development, including their characteristics and challenges that came up in clinical trials with these new biological entities (NBEs). Emphasis is placed on the current view of common side effects observed with passive immunotherapy, so-called amyloid-related imaging abnormalities (ARIAs), and potential ways to overcome this issue. Among these new ideas, a special focus is placed on molecules that are directed against post-translationally modified variants of the Aß peptide, an emerging approach for development of new antibody molecules.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/antagonistas & inibidores , Anticorpos Monoclonais/uso terapêutico , Doença de Alzheimer/diagnóstico , Doença de Alzheimer/imunologia , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/imunologia , Peptídeos beta-Amiloides/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Ensaios Clínicos como Assunto , Citotoxicidade Imunológica , Diagnóstico por Imagem , Modelos Animais de Doenças , Descoberta de Drogas , Humanos , Imunoterapia , Placa Amiloide/tratamento farmacológico , Placa Amiloide/imunologia , Placa Amiloide/metabolismo , Processamento de Proteína Pós-TraducionalRESUMO
Numerous studies suggest that the majority of amyloid-ß (Aß) peptides deposited in Alzheimer's disease (AD) are truncated and post-translationally modified at the N terminus. Among these modified species, pyroglutamyl-Aß (pE-Aß, including N3pE-Aß40/42 and N11pE-Aß40/42) has been identified as particularly neurotoxic. The N-terminal modification renders the peptide hydrophobic, accelerates formation of oligomers, and reduces degradation by peptidases, leading ultimately to the accumulation of the peptide and progression of AD. It has been shown that the formation of pyroglutamyl residues is catalyzed by glutaminyl cyclase (QC). Here, we present data about the pharmacological in vitro and in vivo efficacy of the QC inhibitor (S)-1-(1H-benzo[d]imidazol-5-yl)-5-(4-propoxyphenyl)imidazolidin-2-one (PQ912), the first-in-class compound that is in clinical development. PQ912 inhibits human, rat, and mouse QC activity, with Ki values ranging between 20 and 65 nM. Chronic oral treatment of hAPPSLxhQC double-transgenic mice with approximately 200 mg/kg/day via chow shows a significant reduction of pE-Aß levels and concomitant improvement of spatial learning in a Morris water maze test paradigm. This dose results in a brain and cerebrospinal fluid concentration of PQ912 which relates to a QC target occupancy of about 60%. Thus, we conclude that >50% inhibition of QC activity in the brain leads to robust treatment effects. Secondary pharmacology experiments in mice indicate a fairly large potency difference for Aß cyclization compared with cyclization of physiologic substrates, suggesting a robust therapeutic window in humans. This information constitutes an important translational guidance for predicting the therapeutic dose range in clinical studies with PQ912.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Aminoaciltransferases/antagonistas & inibidores , Benzimidazóis/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Imidazolinas/uso terapêutico , Nootrópicos/uso terapêutico , Doença de Alzheimer/psicologia , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/efeitos dos fármacos , Animais , Benzimidazóis/líquido cefalorraquidiano , Benzimidazóis/farmacocinética , Sítios de Ligação , Ciclização , Sistemas de Liberação de Medicamentos , Inibidores Enzimáticos/líquido cefalorraquidiano , Inibidores Enzimáticos/farmacocinética , Feminino , Células HEK293 , Humanos , Imidazolinas/líquido cefalorraquidiano , Imidazolinas/farmacocinética , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Nootrópicos/líquido cefalorraquidiano , Nootrópicos/farmacocinética , Ligação Proteica , Ratos , Aprendizagem Espacial/efeitos dos fármacosRESUMO
Recombinant expression and purification of amyloid peptides represents a common basis for investigating the molecular mechanisms of amyloid formation and toxicity. However, the isolation of the recombinant peptides is hampered by inefficient separation from contaminants such as the fusion protein required for efficient expression in E. coli. Here, we present a new approach for the isolation of highly purified Aß(1-42) and pGlu-Aß(3-42), which is based on a separation using preparative SDS-PAGE. The method relies on the purification of the Aß fusion protein by affinity chromatography followed by preparative SDS-PAGE under reducing conditions and subsequent removal of detergents by precipitation. The application of preparative SDS-PAGE represents the key step to isolate highly pure recombinant Aß, which has been applied for characterization of aggregation and toxicity. Thereby, the yield of the purification strategy was >60%. To the best of our knowledge, this is the first description of an electrophoresis-based method for purification of a recombinant Aß peptide. Therefore, the method might be of interest for isolation of other amyloid peptides, which are critical for conventional purification strategies due to their aggregation propensity.
Assuntos
Peptídeos beta-Amiloides/isolamento & purificação , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Peptídeos beta-Amiloides/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Humanos , Espectrometria de Massas , Camundongos , Microscopia Eletrônica de Transmissão , Neurônios/citologia , Fragmentos de Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
The astacin proteases meprin α and ß are emerging drug targets for treatment of disorders such as kidney failure, fibrosis or inflammatory bowel disease. However, there are only few inhibitors of both proteases reported to date. Starting from NNGH as lead structure, a detailed elaboration of the structure-activity relationship of meprin ß inhibitors was performed, leading to compounds with activities in the lower nanomolar range. Considering the preference of meprin ß for acidic residues in the P1' position, the compounds were optimized. Acidic modifications induced potent inhibition and >100-fold selectivity over other structurally related metalloproteases such as MMP-2 or ADAM10.
Assuntos
Ácidos Hidroxâmicos/química , Metaloendopeptidases/antagonistas & inibidores , Inibidores de Proteases/química , Sulfonamidas/química , Ácidos Hidroxâmicos/síntese química , Inibidores de Metaloproteinases de Matriz/síntese química , Inibidores de Metaloproteinases de Matriz/química , Inibidores de Proteases/síntese química , Relação Estrutura-Atividade , Sulfonamidas/síntese químicaRESUMO
Recently, Aß peptide variants with an N-terminal truncation and pyroglutamate modification were identified and shown to be highly neurotoxic and prone to aggregation. This modification of Aß is catalyzed by glutaminyl cyclase (QC) and pharmacological inhibition of QC diminishes Aß deposition and accompanying gliosis and ameliorates memory impairment in transgenic mouse models of Alzheimer's disease (AD). QC expression was initially described in the hypothalamus, where thyrotropin-releasing hormone (TRH) is one of its physiological substrates. In addition to its hormonal role, a novel neuroprotective function of TRH following excitotoxicity and Aß-mediated neurotoxicity has been reported in the hippocampus. Functionally matching this finding, we recently demonstrated QC expression by hippocampal interneurons in mouse brain. Here, we detected neuronal co-expression of QC and TRH in the hippocampus of young adult wild type mice using double immunofluorescence labeling. This provides evidence for TRH being a physiological QC substrate in hippocampus. Additionally, in neocortex of aged but not of young mice transgenic for amyloid precursor protein an increase of QC mRNA levels was found compared to wild type littermates. This phenomenon was not observed in hippocampus, which is later affected by Aß pathology. However, in hippocampus of transgenic - but not of wild type mice - a correlation between QC and TRH mRNA levels was revealed. This co-regulation of the enzyme QC and its substrate TRH was reflected by a co-induction of both proteins in reactive astrocytes in proximity of Aß deposits. Also, in primary mouse astrocytes a co-induction of QC and TRH was demonstrated upon Aß stimulation.
Assuntos
Aminoaciltransferases/metabolismo , Astrócitos/enzimologia , Hipocampo/enzimologia , Neurônios/enzimologia , Hormônio Liberador de Tireotropina/metabolismo , Aminoaciltransferases/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Sequência de Bases , Primers do DNA , Hipocampo/citologia , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , Especificidade por Substrato , Hormônio Liberador de Tireotropina/genéticaRESUMO
Secretory peptides and proteins are frequently modified by pyroglutamic acid (pE, pGlu) at their N-terminus. This modification is catalyzed by the glutaminyl cyclases QC and isoQC. Here, we decipher the roles of the isoenzymes by characterization of IsoQC-/- mice. These mice show a significant reduction of glutaminyl cyclase activity in brain and peripheral tissue, suggesting ubiquitous expression of the isoQC enzyme. An assay of substrate conversion in vivo reveals impaired generation of the pGlu-modified C-C chemokine ligand 2 (CCL2, MCP-1) in isoQC-/- mice. The pGlu-formation was also impaired in primary neurons, which express significant levels of QC. Interestingly, however, the formation of the neuropeptide hormone thyrotropin-releasing hormone (TRH), assessed by immunohistochemistry and hormonal analysis of hypothalamic-pituitary-thyroid axis, was not affected in isoQC-/-, which contrasts to QC-/-. Thus, the results reveal differential functions of isoQC and QC in the formation of the pGlu-peptides CCL2 and TRH. Substrates requiring extensive prohormone processing in secretory granules, such as TRH, are primarily converted by QC. In contrast, protein substrates such as CCL2 appear to be primarily converted by isoQC. The results provide a new example, how subtle differences in subcellular localization of enzymes and substrate precursor maturation might influence pGlu-product formation.
Assuntos
Aminoaciltransferases/metabolismo , Administração Oral , Aminoaciltransferases/deficiência , Animais , Células Cultivadas , Glucose/administração & dosagem , Teste de Tolerância a Glucose , Inflamação/induzido quimicamente , Inflamação/metabolismo , Isoenzimas/metabolismo , Lipopolissacarídeos/administração & dosagem , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Ácido Pirrolidonocarboxílico/metabolismo , Especificidade por SubstratoRESUMO
The brains of Alzheimer's disease (AD) patients are characterized by deposits of Abeta peptides and by accompanying chronic inflammation. Here, we provide evidence that the enzyme isoglutaminyl cyclase (isoQC) is a novel factor contributing to both aspects of AD pathology. Two putative substrates of isoQC, N-truncated Abeta peptides and the monocyte chemoattractant chemokine CCL2, undergo isoQC-catalyzed pyroglutamate (pGlu) modification. This triggers Abeta aggregation and facilitates the biological activity of CCL2, which collectively results in the formation of high molecular weight Abeta aggregates, glial cell activation, neuroinflammation and neuronal cell death. In mouse brain, we found isoQC to be neuron-specifically expressed in neocortical, hippocampal and subcortical structures, localized to the endoplasmic reticulum and Golgi apparatus as well as co-expressed with its substrate CCL2. In aged APP transgenic Tg2576 mice, both isoQC and CCL2 mRNA levels are up-regulated and isoQC and CCL2 proteins were found to be co-induced in Abeta plaque-associated reactive astrocytes. Also, in mouse primary astrocyte culture, a simultaneous up-regulation of isoQC and CCL2 expression was revealed upon Abeta and pGlu-Abeta stimulation. In brains of AD patients, the expression of isoQC and CCL2 mRNA and protein is up-regulated compared to controls and correlates with pGlu-Abeta load and with the decline in mini-mental state examination. Our observations provide evidence for a dual involvement of isoQC in AD pathogenesis by catalysis of pGlu-Abeta and pGlu-CCL2 formation which mutually stimulate inflammatory events and affect cognition. We conclude that isoQC inhibition may target both major pathological events in the development of AD.
Assuntos
Doença de Alzheimer/patologia , Aminoaciltransferases/metabolismo , Encéfalo/metabolismo , Quimiocina CCL2/metabolismo , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Aminoaciltransferases/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Encéfalo/patologia , Células Cultivadas , Quimiocina CCL2/genética , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Fatores de Tempo , Regulação para Cima/genéticaRESUMO
Phosphate ions and glutaminyl cyclase (QC) both catalyze the formation of pyroglutamate (pE, pGlu) from N-terminal glutamine residues of peptides and proteins. Here, we studied the mechanism of glutamine cyclization using kinetic secondary deuterium and solvent isotope effects. The data suggest that proton transfer(s) are rate determining for the spontaneous reaction, and that phosphate and QC are accelerating the reaction by promoting synchronized proton transfers in a concerted mechanism. Thus, non-enzymatic and enzymatic catalysis of pyroglutamate formation exploit a similar mode of transition-state stabilization.