RESUMO
Treatment of cancers in the lung remains a critical challenge in the clinic for which gene therapy could offer valuable options. We describe an effective approach through systemic injection of engineered polymer/DNA nanoparticles that mediate tumor-specific expression of a therapeutic gene, under the control of the cancer-selective progression elevated gene 3 (PEG-3) promoter, to treat tumors in the lungs of diseased mice. A clinically tested, untargeted, polyethylenimine carrier was selected to aid rapid transition to clinical studies, and a CpG-free plasmid backbone and coding sequences were used to reduce inflammation. Intravenous administration of nanoparticles expressing murine single-chain interleukin 12, under the control of PEG-3 promoter, significantly improved the survival of mice in both an orthotopic and a metastatic model of lung cancer with no marked symptoms of systemic toxicity. These outcomes achieved using clinically relevant nanoparticle components raises the promise of translation to human therapy.
Assuntos
DNA/administração & dosagem , Técnicas de Transferência de Genes , Terapia Genética , Interleucina-12/genética , Neoplasias Pulmonares/terapia , Animais , DNA/genética , DNA/uso terapêutico , Modelos Animais de Doenças , Expressão Gênica , Humanos , Injeções , Neoplasias Pulmonares/genética , Camundongos , Camundongos SCID , Nanomedicina , Nanopartículas/administração & dosagem , Nanopartículas/química , Polietilenoimina/administração & dosagem , Polietilenoimina/químicaRESUMO
Inhibition of the NGF/TrkA interaction presents an interesting alternative to the use of non-steroidal anti-inflammatories and/or opioids for the control of inflammatory, chronic and neuropathic pain. Most prominent of the current approaches to this therapy is the antibody Tanezumab, which is a late-stage development humanized monoclonal antibody that targets NGF. We sought to determine whether peptides might similarly inhibit the NGF/TrkA interaction and so serve as future therapeutic leads. Starting from two peptides that inhibit the NGF/TrkA interaction, we sought to eliminate a cysteine residue close to the C-terminal of both sequences, by an approach of mutagenic analysis and saturation mutagenesis of mutable residues. Elimination of cysteine from a therapeutic lead is desirable to circumvent manufacturing difficulties resulting from oxidation. Our analyses determined that the cysteine residue is not required for NGF binding, but is essential for inhibition of the NGF/TrkA interaction at pharmacologically relevant peptide concentrations. We conclude that a cysteine residue is required within potential peptide-based therapeutic leads and hypothesise that these peptides likely act as dimers, mirroring the dimeric structure of the TrkA receptor.
Assuntos
Cisteína/química , Biblioteca de Peptídeos , Inibidores de Proteínas Quinases/química , Receptor trkA , Anticorpos Monoclonais Humanizados/química , Humanos , Receptor trkA/antagonistas & inibidores , Receptor trkA/químicaRESUMO
Molecular imaging of cancers using probes specific for tumor-associated target proteins offers a powerful solution for providing information regarding selection of targeted therapy, patient stratification, and response to therapy. Here we demonstrate the power of bicyclic peptides as targeting probes, exemplified with the tumor-overexpressed matrix metalloproteinase MT1-MMP as a target. A bicyclic peptide with subnanomolar affinity towards MT1-MMP was identified, and its radioconjugate showed selective tumor uptake in an HT1080 xenograft mouse model. Proteolytic stabilization of the peptide by chemical modification significantly enhanced the in vivo tumor signal [from 2.5%ID/g to 12%ID/g at 1 hour post injection (p.i.)]. Studies using mouse xenograft models with different cell lines show a robust correlation between tumor signals and in vivo MT1-MMP expression levels. Fatty acid modification of the bicyclic peptide extended its circulating half-life, resulting in increased tumor signals (36%ID/g at 6 hours p.i.). Comparative work with an equipotent radiolabeled MT1-MMP targeting antibody demonstrated starkly differential biodistribution and tumor accumulation properties, with the tumor signal slowly increasing to 6.2%ID/g within 48 hours. The rapid tumor penetration characteristics of bicyclic peptides, coupled with high potency and chemical versatility, thus offer high-contrast imaging probes for clinical diagnostics with compelling additional potential in targeted therapy.Significance: This work demonstrates the potential of bicyclic peptides as a platform for the development of high-contrast imaging probes for potential use in clinical cancer diagnostics and molecularly targeted therapeutics.
Assuntos
Anticorpos Monoclonais/farmacologia , Inibidores Enzimáticos/farmacologia , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Metaloproteinase 14 da Matriz/química , Peptídeos Cíclicos/farmacologia , Animais , Anticorpos Monoclonais/farmacocinética , Apoptose , Proliferação de Células , Inibidores Enzimáticos/farmacocinética , Fibrossarcoma/diagnóstico por imagem , Fibrossarcoma/patologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Processamento de Imagem Assistida por Computador/métodos , Masculino , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Peptídeos Cíclicos/farmacocinética , Tomografia por Emissão de Pósitrons , Distribuição Tecidual , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Plasma kallikrein, a member of the kallikrein-kinin system, catalyzes the release of the bioactive peptide bradykinin, which induces inflammation, vasodilation, vessel permeability, and pain. Preclinical evidence implicates the activity of plasma kallikrein in diabetic retinopathy, which is a leading cause of visual loss in patients suffering from diabetes mellitus. Employing a technology based on phage-display combined with chemical cyclization, we have identified highly selective bicyclic peptide inhibitors with nano- and picomolar potencies toward plasma kallikrein. Stability in biological matrices was either intrinsic to the peptide or engineered via the introduction of non-natural amino acids and nonpeptidic bonds. The peptides prevented bradykinin release in vitro, and in vivo efficacy was demonstrated in both a rat paw edema model and in rodent models of diabetes-induced retinal permeability. With a highly extended half-life of â¼40 h in rabbit eyes following intravitreal administration, the bicyclic peptides are promising novel agents for the treatment of diabetic retinopathy and diabetic macular edema.
Assuntos
Compostos Bicíclicos com Pontes/síntese química , Compostos Bicíclicos com Pontes/farmacologia , Complicações do Diabetes/tratamento farmacológico , Retinopatia Diabética/tratamento farmacológico , Edema Macular/tratamento farmacológico , Edema Macular/etiologia , Calicreína Plasmática/antagonistas & inibidores , Inibidores de Proteases/síntese química , Inibidores de Proteases/farmacologia , Animais , Bradicinina/metabolismo , Edema/tratamento farmacológico , Olho/metabolismo , Pé/patologia , Meia-Vida , Injeções Intravítreas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Permeabilidade , Inibidores de Proteases/administração & dosagem , Coelhos , Ratos , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Especificidade por Substrato , Corpo Vítreo/química , Corpo Vítreo/metabolismoRESUMO
Phosphatidic acid and phosphatidylserine are negatively charged abundant phospholipids with well-recognized structural roles in cellular membranes. They are also signaling lipids since their regulated formation (or appearance) can constitute an important signal for downstream responses. The list of potential effectors for these lipids is expanding rapidly and includes proteins involved in virtually all aspects of cellular regulation. Because it is not always clear whether these effectors recognize the specific phospholipids or a general negatively-charged membrane environment, questions about specificity must be addressed on a case by case basis. In this review we present an up to date list of potential phosphatidic acid- and phosphatidylserine-binding proteins.
Assuntos
Proteínas de Transporte/química , Membrana Celular/química , Membranas Intracelulares/química , Ácidos Fosfatídicos/química , Fosfatidilserinas/química , Sequência de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Membrana Celular/metabolismo , Humanos , Membranas Intracelulares/metabolismo , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Molecular , Ácidos Fosfatídicos/metabolismo , Fosfatidilserinas/metabolismo , Transdução de SinaisRESUMO
The adaptor protein Gab-2 coordinates the assembly of the IL-3 signalsome comprising Gab-2, Grb2, Shc, SHP-2 and PI3K. To investigate the role of the pleckstrin homology domain of Gab-2 in this process, epitope-tagged wild type Gab-2 (WTGab-2), Gab-2 lacking its PH domain (DeltaPHGab-2) and the Gab-2 PH domain alone (PHGab-2) were inducibly expressed in IL-3-dependent BaF/3 cells. Expression of DeltaPHGab-2 reduced IL-3-dependent proliferation and long-term activation of ERK1 and 2 and PKB by IL-3. While we demonstrate that the Gab-2 PH domain can bind PI(3,4,5)P3, it is dispensable for Gab-2 membrane localisation, tyrosine phosphorylation and signalsome formation. Rather, the proline-rich motifs of Gab-2 appear to contribute to the constitutive membrane localisation we observe, independently of tyrosine phosphorylation or the PH domain. Taken together, these findings suggest that once Gab-2 is tyrosine phosphorylated its PH domain is required for the optimal stabilisation of the signalsome, enabling full activation of downstream signals.