RESUMO
It is assumed that amyloid-ß aggregation is a crucial event in the pathogenesis of Alzheimer's disease. Novel 2,6-disubstituted pyridine derivatives were designed to interact with the ß-sheet conformation of Aß via donor-acceptor-donor hydrogen bond formation. A series of pyridine derivatives were synthesized and tested regarding their potential to inhibit the aggregation of Aß. The 2,6-diaminopyridine moiety was identified as a key component to inhibit Aß aggregation. Overall, compounds having three 2,6-disubstituted pyridine units separated by at least one C2- or C3-linker displayed the most potent inhibition of Aß aggregation.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Piridinas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Agregados Proteicos/efeitos dos fármacos , Piridinas/síntese química , Piridinas/química , Relação Estrutura-AtividadeRESUMO
In Down syndrome (DS) or trisomy of chromosome 21, the ß-amyloid (Aß) peptide product of the amyloid precursor protein (APP) is present in excess. Evidence points to increased APP gene dose and Aß as playing a critical role in cognitive difficulties experienced by people with DS. Particularly, Aß is linked to the late-life emergence of dementia as associated with neuropathological markers of Alzheimer's disease (AD). At present, no treatment targets Aß-related pathogenesis in people with DS. Herein we used a vaccine containing the Aß 1-15 peptide embedded into liposomes together with the adjuvant monophosphoryl lipid A (MPLA). Ts65Dn mice, a model of DS, were immunized with the anti-Aß vaccine at 5 months of age and were examined for cognitive measures at 8 months of age. The status of basal forebrain cholinergic neurons and brain levels of APP and its proteolytic products were measured. Immunization of Ts65Dn mice resulted in robust anti-Aß IgG titers, demonstrating the ability of the vaccine to break self-tolerance. The vaccine-induced antibodies reacted with Aß without detectable binding to either APP or its C-terminal fragments. Vaccination of Ts65Dn mice resulted in a modest, but non-significant reduction in brain Aß levels relative to vehicle-treated Ts65Dn mice, resulting in similar levels of Aß as diploid (2N) mice. Importantly, vaccinated Ts65Dn mice showed resolution of memory deficits in the novel object recognition and contextual fear conditioning tests, as well as reduction of cholinergic neuron atrophy. No treatment adverse effects were observed; vaccine did not result in inflammation, cellular infiltration, or hemorrhage. These data are the first to show that an anti-Aß immunotherapeutic approach may act to target Aß-related pathology in a mouse model of DS.
Assuntos
Peptídeos beta-Amiloides/imunologia , Transtornos Cognitivos/complicações , Transtornos Cognitivos/tratamento farmacológico , Síndrome de Down/complicações , Síndrome de Down/tratamento farmacológico , Vacinas/uso terapêutico , Peptídeos beta-Amiloides/genética , Animais , Animais Recém-Nascidos , Anticorpos/metabolismo , Atrofia , Comportamento Animal , Biomarcadores/metabolismo , Encéfalo/metabolismo , Encéfalo/patologia , Neurônios Colinérgicos/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Hemorragia/patologia , Inflamação/patologia , Masculino , Memória , Camundongos Transgênicos , Núcleos Septais/patologia , VacinaçãoRESUMO
Immunoglobulin class switching from IgM to IgG in response to peptides is generally T cell-dependent and vaccination in T cell-deficient individuals is inefficient. We show that a vaccine consisting of a dense array of peptides on liposomes induced peptide-specific IgG responses totally independent of T-cell help. Independency was confirmed in mice lacking T cells and in mice deficient for MHC class II, CD40L, and CD28. The IgG titers were high, long-lived, and comparable with titers obtained in wild-type animals, and the antibody response was associated with germinal center formation, expression of activation-induced cytidine deaminase, and affinity maturation. The T cell-independent (TI) IgG response was strictly dependent on ligation of TLR4 receptors on B cells, and concomitant TLR4 and cognate B-cell receptor stimulation was required on a single-cell level. Surprisingly, the IgG class switch was mediated by TIR-domain-containing adapter inducing interferon-ß (TRIF), but not by MyD88. This study demonstrates that peptides can induce TI isotype switching when antigen and TLR ligand are assembled and appropriately presented directly to B lymphocytes. A TI vaccine could enable efficient prophylactic and therapeutic vaccination of patients with T-cell deficiencies and find application in diseases where induction of T-cell responses contraindicates vaccination, for example, in Alzheimer disease.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/fisiologia , Peptídeos beta-Amiloides/imunologia , Linfócitos B/imunologia , Switching de Imunoglobulina/imunologia , Fragmentos de Peptídeos/imunologia , Receptor 4 Toll-Like/fisiologia , Vacinas de Subunidades Antigênicas/imunologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Transferência Adotiva , Sequência de Aminoácidos , Peptídeos beta-Amiloides/administração & dosagem , Animais , Apresentação de Antígeno , Linfócitos B/metabolismo , Antígenos CD28/deficiência , Antígenos CD28/imunologia , Ligante de CD40/deficiência , Ligante de CD40/imunologia , Centro Germinativo/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina M/biossíntese , Receptores de Lipopolissacarídeos/imunologia , Lipossomos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Dados de Sequência Molecular , Ovalbumina/administração & dosagem , Ovalbumina/imunologia , Fragmentos de Peptídeos/administração & dosagem , Receptores de Antígenos de Linfócitos B/imunologia , Linfócitos T/imunologia , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genética , Vacinação , Vacinas de Subunidades Antigênicas/administração & dosagemRESUMO
Increasing evidence implicates Aß peptides self-assembly and fibril formation as crucial events in the pathogenesis of Alzheimer disease. Thus, inhibiting Aß aggregation, among others, has emerged as a potential therapeutic intervention for this disorder. Herein, we employed 3-aminopyrazole as a key fragment in our design of non-dye compounds capable of interacting with Aß42 via a donor-acceptor-donor hydrogen bond pattern complementary to that of the ß-sheet conformation of Aß42. The initial design of the compounds was based on connecting two 3-aminopyrazole moieties via a linker to identify suitable scaffold molecules. Additional aryl substitutions on the two 3-aminopyrazole moieties were also explored to enhance π-π stacking/hydrophobic interactions with amino acids of Aß42. The efficacy of these compounds on inhibiting Aß fibril formation and toxicity in vitro was assessed using a combination of biophysical techniques and viability assays. Using structure activity relationship data from the in vitro assays, we identified compounds capable of preventing pathological self-assembly of Aß42 leading to decreased cell toxicity.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Peptídeos beta-Amiloides/química , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/química , Pirazóis/química , Linhagem Celular Tumoral , Citotoxinas/antagonistas & inibidores , Citotoxinas/química , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Estrutura Secundária de Proteína , Relação Estrutura-AtividadeRESUMO
Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule ß-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing ß-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized ß-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.
Assuntos
Lipossomos/química , Peptídeos/química , Deficiências na Proteostase/metabolismo , Vacinas/química , Doença de Alzheimer/metabolismo , Animais , Benzotiazóis , Dicroísmo Circular , Feminino , Humanos , Imunoglobulina G/química , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Endogâmicos C57BL , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Tiazóis/químicaRESUMO
We investigated the therapeutic effects of two different versions of Abeta(1-15 (16)) liposome-based vaccines. Inoculation of APP-V717IxPS-1 (APPxPS-1) double-transgenic mice with tetra-palmitoylated amyloid 1-15 peptide (palmAbeta(1-15)), or with amyloid 1-16 peptide (PEG-Abeta(1-16)) linked to a polyethyleneglycol spacer at each end, and embedded within a liposome membrane, elicited fast immune responses with identical binding epitopes. PalmAbeta(1-15) liposomal vaccine elicited an immune response that restored the memory defect of the mice, whereas that of PEG-Abeta(1-16) had no such effect. Immunoglobulins that were generated were predominantly of the IgG class with palmAbeta(1-15), whereas those elicited by PEG-Abeta(1-16) were primarily of the IgM class. The IgG subclasses of the antibodies generated by both vaccines were mostly IgG2b indicating noninflammatory Th2 isotype. CD and NMR revealed predominantly beta-sheet conformation of palmAbeta(1-15) and random coil of PEG-Abeta(1-16). We conclude that the association with liposomes induced a variation of the immunogenic structures and thereby different immunogenicities. This finding supports the hypothesis that Alzheimer's disease is a "conformational" disease, implying that antibodies against amyloid sequences in the beta-sheet conformation are preferred as potential therapeutic agents.