RESUMO
Aptamers may form well-defined three-dimensional structures binding with high affinity and stability to a specific receptor. The aptamer anti-MUC1 isoform Y is one the most used due the affinity to MUC1, which is overexpressed in several types of cancer and inflammation process. In this study we have developed, characterized, in vitro as in vivo evaluated a nanoaptamer (anti-MUC1/Y) as a nanoagent for rheumatoid arthritis treatment. The results showed that a nanoaptamer with a size range of 241 nm was produced. The entrapment efficacy was 90% with a biodistribution showing a high hepatic uptake (>98%). The results in vivo showed a potent effect in arthritis experimental model, especially in low doses. The results corroborate the applicability of this nanosystem for RA treatment.
Assuntos
Aptâmeros de Nucleotídeos , Artrite , Nanopartículas , Aptâmeros de Nucleotídeos/química , Humanos , Mucina-1/química , Nanopartículas/química , Distribuição TecidualRESUMO
The synthesis of MUC1 glycopeptides bearing modified tumor-associated carbohydrate antigens (TACAs) represents an effective strategy to develop potential antitumor vaccines that trigger strong immune response. In this context, we present herein the multistep synthesis of the triazole glycosyl amino acid Neu5Ac-α/ß2-triazole-6-ßGalNAc-ThrOH 1 as STn antigen analog, along with its assembly on the corresponding MUC1 peptide to give NAcProAsp [Neu5Acα/ß2-triazole-6-ßGalNAc]ThrArgProGlyOH 2. Despite interacting differently with SM3 monoclonal antibody, as shown by molecular dynamic simulations, this unnatural triazole glycopeptide may represent a promising candidate for cancer immunotherapy.
Assuntos
Antígenos Glicosídicos Associados a Tumores/química , Glicopeptídeos/química , Glicopeptídeos/síntese química , Mucina-1/química , Triazóis/química , Técnicas de Química SintéticaRESUMO
SEA domains are ubiquitous in large proteins associated with highly glycosylated environments. Certain SEA domains undergo intramolecular proteolysis involving a nucleophilic attack of a serine hydroxyl group on the preceding glycine carbonyl. The mucin-1 (MUC1) SEA domain has been extensively investigated as a model of intramolecular proteolysis. Since neither a general base, a general acid, nor an oxyanion hole could be identified in MUC1 SEA, it has been suggested that proteolysis is accelerated by a non-planarity of the scissile peptide bond imposed by protein folding. A reactant distorted peptide bond has been also invoked to explain the autoproteolysis of several unrelated proteins. However, the only evidence of peptide distortion in MUC1 SEA stems from molecular dynamic simulations of the reactant modeled upon a single NMR structure of the cleaved product. We report the first high-resolution X-ray structure of cleaved MUC1 SEA. Structural comparison with uncleaved SEA domains suggests that the number of residues evolutionarily inserted in the cleaved loop of MUC1 SEA precludes the formation of a properly hydrogen-bonded beta turn. By sequence analysis, we show that this conformational frustration is shared by all known cleaved SEA domains. In addition, alternative conformations of the uncleaved precursor could be modeled in which the scissile peptide bond is planar. The implications of these structures for autoproteolysis are discussed in the light of the previous research on autoproteolysis.
Assuntos
Mucina-1/química , Cristalografia por Raios X , Modelos Moleculares , Mucina-1/metabolismo , Domínios Proteicos , ProteóliseRESUMO
The use of monoclonal antibodies and aptamers is growing every single day, as the use of nanoparticle systems. Although most of the products are under investigation, there are a few commercialized products available at the market, for human consume. In this study, we have compared three formulations (aptamer anti-MUC1, monoclonal antibody - Trastuzumab and monoclonal antibodies nanoparticles - PLA/PVA/MMT trastuzumab) to identify their profile as also to understand their behavior into an alive biological system. In this direction the radiolabeling of the products were done and they were all tested in animals (in vivo) in two conditions: healthy rats and breast cancer induced animals. The results showed that the nanoparticle has the better biodistribution profile, followed by the aptamer. We conclude that more studies and a global effort to elucidate the biological behavior of drugs and especially nano-drugs are necessary.
Assuntos
Antineoplásicos/farmacocinética , Aptâmeros de Peptídeos/farmacocinética , Glândulas Mamárias Animais/diagnóstico por imagem , Neoplasias Mamárias Experimentais/diagnóstico por imagem , Cintilografia/métodos , Trastuzumab/farmacocinética , Animais , Antineoplásicos/química , Aptâmeros de Peptídeos/química , Feminino , Humanos , Glândulas Mamárias Animais/metabolismo , Glândulas Mamárias Animais/patologia , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Mucina-1/química , Mucina-1/metabolismo , Nanopartículas/química , Nanopartículas/metabolismo , Poliésteres/química , Álcool de Polivinil/química , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Wistar , Coloração e Rotulagem/métodos , Tecnécio/química , Tecnécio/farmacocinética , Distribuição Tecidual , Trastuzumab/químicaRESUMO
This study presents the synthesis of the novel protected O-glycosylated amino acid derivatives 1 and 2, containing ßGalNAc-SerOBn and ßGalNAc-ThrOBn units, respectively, as mimetics of the natural Tn antigen (αGalNAc-Ser/Thr), along with the solid-phase assembly of the glycopeptides NHAcSer-Ala-Pro-Asp-Thr[αGalNAc]-Arg-Pro-Ala-Pro-Gly-BSA (3-BSA) and NHAcSer-Ala-Pro-Asp-Thr[ßGalNAc]-Arg-Pro-Ala-Pro-Gly-BSA (4-BSA), bearing αGalNAc-Thr or ßGalNAc-Thr units, respectively, as mimetics of MUC1 tumor mucin glycoproteins. According to ELISA tests, immunizations of mice with ßGalNAc-glycopeptide 4-BSA induced higher sera titers (1:320 000) than immunizations with αGalNAc-glycopeptide 3-BSA (1:40 000). Likewise, flow cytometry assays showed higher capacity of the obtained anti-glycopeptide 4-BSA antibodies to recognize MCF-7 tumor cells. Cross-recognition between immunopurified anti-ßGalNAc antibodies and αGalNAc-glycopeptide and vice versa was also verified. Lastly, molecular dynamics simulations and surface plasmon resonance (SPR) showed that ßGalNAc-glycopeptide 4 can interact with a model antitumor monoclonal antibody (SM3). Taken together, these data highlight the improved immunogenicity of the unnatural glycopeptide 4-BSA, bearing ßGalNAc-Thr as Tn antigen isomer.
Assuntos
Anticorpos Monoclonais/metabolismo , Formação de Anticorpos/efeitos dos fármacos , Antígenos Glicosídicos Associados a Tumores/química , Mucina-1/metabolismo , Mucina-1/farmacologia , Animais , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Bioensaio/normas , Sequência de Carboidratos , Humanos , Isomerismo , Células MCF-7 , Camundongos , Modelos Biológicos , Simulação de Dinâmica Molecular , Mucina-1/química , Técnicas de Síntese em Fase Sólida , Ressonância de Plasmônio de SuperfícieRESUMO
Inversions of short genomic sequences may play a central role in the generation of protein complexity. We report here the existence of an heterogeneous group of proteins (the trefoil precursors MUC-1 and MUA-1, six preproendothelins, and five classes of zinc finger knot proteins) having both cysteine signatures (Cs) and their inverse complementary sequences (Cs) in the same polypeptide chain. We have also found cases in which the (Cs) of a given signature is not present in the same protein, but elsewhere. TGEKPYK, a cysteine-free motif of the human transcription factor, Krab, coexists with its inverse complementary sequence in 31 proteins; the inverse complementary alone is present in a great number of proteins. Our findings suggest that short DNA inversions are a widespread feature of the genome.
Assuntos
Cisteína , Proteínas/química , Proteínas/genética , Sequência de Aminoácidos , DNA/química , Endotelina-1 , Endotelinas/química , Endotelinas/genética , Humanos , Dados de Sequência Molecular , Mucina-1/química , Mucina-1/genética , Precursores de Proteínas/química , Precursores de Proteínas/genética , Homologia de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transposases/química , Transposases/genética , Dedos de ZincoRESUMO
Breast mucins are expressed by malignant epithelial cells and they elicit an immune reaction. The up-regulation of mucin expression is association with tumour invasion, this mucin called MUC-1 reduces the cell-cell interaction facilitating cell detachment. The MUC-1 gene product, known as polymorphic epithelial mucin is a transmembrane high molecular weight glycoprotein. The molecule of MUC-1 has a central polypeptidic core with a carbohydrate linked in O-linkage to serines and threonines. The carbohydrate side chain epitope of MUC-1 molecule produced by breast cancer cells is heavily sialylated, giving their physical properties and increasing their immunogenicity. The development of monoclonal antibodies (MAb) has led to study the MUC-1 in subcellular extracts, tissues and culture supernatants from breast cancer and also colorectal carcinoma. The pattern of tumour cell staining with labeled MAb varies according with the grade of malignancy; these MAb bind either to peptide sequence and/or to the glycosylated epitopes. MUC-1 has a clinical relevance because serum concentrations may be useful for monitoring the response to therapy and progress of disease. MUC-1 epitope masking has been described since specific antibodies can combine with them forming immune complexes. Finally, mucins have been considered to develop vaccines against cancer, targeting specific carbohydrate and mucin epitopes.