RESUMO
The application of small peptides targeting amyloid beta (Aß) is one of many drug development strategies for the treatment of Alzheimer's disease (AD). We have previously identified several peptides consisting solely of D-enantiomeric amino acid residues obtained from mirror-image phage display selection, which bind to Aß in different assembly states and eliminate toxic Aß aggregates. Some of these D-peptides show both diagnostic and therapeutic potential in vitro and in vivo. Here we have analysed the similarity of the arginine-rich D-peptide D3 to the arginine-rich motif (ARM) of the human immunodeficiency virus type 1 transactivator of transcription (HIV-Tat) protein, and examined its in vivo blood-brain barrier (BBB) permeability using wild type mice and transgenic mouse models of Alzheimer's disease. We are able to demonstrate that D3 rapidly enters the brain where it can be found associated with amyloid plaques suggesting a direct penetration of BBB.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Barreira Hematoencefálica/efeitos dos fármacos , Fármacos Neuroprotetores/farmacocinética , Oligopeptídeos/metabolismo , Administração Oral , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Sequência de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Permeabilidade Capilar , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/metabolismo , Oligopeptídeos/síntese química , Oligopeptídeos/farmacocinética , Alinhamento de Sequência , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
Fluorescence-activated droplet sorting (FADS) is a widely used microfluidic technique for high-throughput screening. However, it requires highly trained specialists to determine optimal sorting parameters, and this results in a large combinatorial space that is challenging to optimize systematically. Additionally, it is currently challenging to track every single droplet within a screen, leading to compromised sorting and "hidden" false-positive events. To overcome these limitations, we have developed a setup in which the droplet frequency, spacing, and trajectory at the sorting junction are monitored in real time using impedance analysis. The resulting data are used to continuously optimize all parameters automatically and to counteract perturbations, resulting in higher throughput, higher reproducibility, increased robustness, and a beginner-friendly character. We believe this provides a missing piece for the spreading of phenotypic single-cell analysis methods, similar to what we have seen for single-cell genomics platforms.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Microfluídica/métodos , Reprodutibilidade dos Testes , Genômica , Análise de Célula Única/métodosRESUMO
Currently, there are no causative or disease modifying treatments available for Alzheimer's disease (AD). Previously, it has been shown that D3, a small, fully d-enantiomeric peptide is able to eliminate low molecular weight Aß oligomers in vitro, enhance cognition and reduce plaque load in AD transgenic mice. To further characterise the therapeutic potential of D3 towards N-terminally truncated and pyroglutamated Aß (pEAß(3-42)) we tested D3 and its head-to-tail tandem derivative D3D3 both in vitro and in vivo in the new mouse model TBA2.1. These mice produce human pEAß(3-42) leading to a strong, early onset motor neurodegenerative phenotype. In the present study, we were able to demonstrate 1) strong binding affinity of both D3 and D3D3 to pEAß(3-42) in comparison to Aß(1-42) and 2) increased affinity of the tandem derivative D3D3 in comparison to D3. Subsequently we tested the therapeutic potentials of both peptides in the TBA2.1 animal model. Truly therapeutic, non-preventive treatment with D3 and D3D3 clearly slowed the progression of the neurodegenerative TBA2.1 phenotype, indicating the strong therapeutic potential of both peptides against pEAß(3-42) induced neurodegeneration.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Cognição/fisiologia , Fragmentos de Peptídeos/metabolismo , Placa Amiloide/metabolismo , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Animais , Modelos Animais de Doenças , Camundongos Transgênicos , Fragmentos de Peptídeos/genética , Fenótipo , Placa Amiloide/genéticaRESUMO
Fluorescence-activated droplet sorting (FADS) has become a widely used technique for high-throughput screening applications. However, existing methods are very sensitive to fluctuating flow rates at the sorting junction, which can be caused by the pulsing effects of mechanical pumps, droplet aggregates or the accumulation of precipitates during lengthy biological screening applications. Furthermore, existing sorting devices allow only 2-way sorting. We present here a dielectrophoretic sorting system in which the droplets are sorted along multiple electrode pairs that run parallel to the channels. This enables highly reliable sorting (no errors were detected for more than 2000 sorting events) even when inverting the relative flow rates at a 2-way sorting junction from 80 : 20 to 20 : 80. Furthermore, our toolbox is scalable: we demonstrate on the example of a triple-colour sorting experiment with a total of four decoupled electrodes that multi-way sorting is feasible.
RESUMO
Alzheimer's disease (AD) is a neurodegenerative disorder and the most common type of dementia. Until now, there is no curative therapy available. Previously, we selected the amyloid-beta (Aß) targeting peptide D3 consisting of 12 d-enantiomeric amino acid residues by mirror image phage display as a potential drug candidate for the treatment of AD. In the current approach, we investigated the optimization potential of linear D3 with free C-terminus (D3COOH) by chemical modifications. First, the impact of the net charge was investigated and second, cyclization was introduced which is a well-known tool for the optimization of peptides for enhanced target affinity. Following this strategy, three D3 derivatives in addition to D3COOH were designed: C-terminally amidated linear D3 (D3CONH2), cyclic D3 (cD3), and cyclic D3 with an additional arginine residue (cD3r) to maintain the net charge of linear D3CONH2. These four compounds were compared to each other according to their binding affinities to Aß(1-42), their efficacy to eliminate cytotoxic oligomers, and consequently their potency to neutralize Aß(1-42) oligomer induced neurotoxicity. D3CONH2 and cD3r versions with equally increased net charge showed superior properties over D3COOH and cD3, respectively. The cyclic versions showed superior properties compared to their linear version with equal net charge, suggesting cD3r to be the most efficient compound among these four. Indeed, treatment of the transgenic AD mouse model Tg-SwDI with cD3r significantly enhanced spatial memory and cognition of these animals as revealed by water maze performance. Therefore, charge increase and cyclization imply suitable modification steps for an optimization approach of the Aß targeting compound D3.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Peptídeos Cíclicos/química , Peptídeos Cíclicos/uso terapêutico , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/ultraestrutura , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular Tumoral , Transtornos Cognitivos/tratamento farmacológico , Transtornos Cognitivos/etiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Conformação Molecular/efeitos dos fármacos , Mutação/genética , Neuroblastoma/patologia , Oligopeptídeos/química , Fragmentos de Peptídeos/ultraestrutura , Peptídeos Cíclicos/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patologia , Placa Amiloide/ultraestrutura , Ligação Proteica/efeitos dos fármacos , EstereoisomerismoRESUMO
Biolayer interferometry is a method to analyze protein interactions in real-time. In this study, we illustrate the usefulness to quantitatively analyze high affinity protein ligand interactions employing a kinetic titration series for characterizing the interactions between two pairs of interaction patterns, in particular immunoglobulin G and protein G B1 as well as scFv IC16 and amyloid beta (1-42). Kinetic titration series are commonly used in surface plasmon resonance and involve sequential injections of analyte over a desired concentration range on a single ligand coated sensor chip without waiting for complete dissociation between the injections. We show that applying this method to biolayer interferometry is straightforward and i) circumvents problems in data evaluation caused by unavoidable sensor differences, ii) saves resources and iii) increases throughput if screening a multitude of different analyte/ligand combinations.
Assuntos
Interferometria/métodos , Imunoglobulina G , Cinética , Ligação Proteica , Ressonância de Plasmônio de SuperfícieRESUMO
There is strong evidence that the amyloid-beta peptide (Aß) plays a central role in the pathogenesis of Alzheimer's disease (AD). In this context, a detailed quantitative description of the interactions with different Aß species is essential for characterization of physiological and artificial ligands. However, the high aggregation propensity of Aß in concert with its susceptibility to structural changes due to even slight changes in solution conditions has impeded surface plasmon resonance (SPR) studies with homogeneous Aß conformer species. Here, we have adapted the experimental procedures to state-of-the-art techniques and established novel approaches to reliably overcome the aforementioned challenges. We show that the application of density gradient centrifugation (DGC) for sample purification and the use of a single chain variable fragment (scFv) of a monoclonal antibody directed against the amino-terminus of Aß allows reliable SPR measurements and quality control of the immobilized Aß aggregate species at any step throughout the experiment.