RESUMO
Dystroglycanopathies are diseases characterized by progressive muscular degeneration and impairment of patient's quality of life. They are associated with altered glycosylation of the dystrophin-glycoprotein (DGC) complex components, such as α-dystroglycan (α-DG), fundamental in the structural and functional stability of the muscle fiber. The diagnosis of dystroglycanopathies is currently based on the observation of clinical manifestations, muscle biopsies and enzymatic measures, and the available monoclonal antibodies are not specific for the dystrophic hypoglycosylated muscle condition. Thus, modified α-DG mucins have been considered potential targets for the development of new diagnostic strategies toward these diseases. In this context, this work describes the synthesis of the hypoglycosylated α-DG mimetic glycopeptide NHAc-Gly-Pro-Thr-Val-Thr[αMan]-Ile-Arg-Gly-BSA (1) as a potential tool for the development of novel antibodies applicable to dystroglycanopathies diagnosis. Glycopeptide 1 was used for the development of polyclonal antibodies and recombinant monoclonal antibodies by Phage Display technology. Accordingly, polyclonal antibodies were reactive to glycopeptide 1, which enables the application of anti-glycopeptide 1 antibodies in immune reactive assays targeting hypoglycosylated α-DG. Regarding monoclonal antibodies, for the first time variable heavy (VH) and variable light (VL) immunoglobulin domains were selected by Phage Display, identified by NGS and described by in silico analysis. The best-characterized VH and VL domains were cloned, expressed in E. coli Shuffle T7 cells, and used to construct a single chain fragment variable that recognized the Glycopeptide 1 (GpαDG1 scFv). Molecular modelling of glycopeptide 1 and GpαDG1 scFv suggested that their interaction occurs through hydrogen bonds and hydrophobic contacts involving amino acids from scFv (I51, Y33, S229, Y235, and P233) and R8 and α-mannose from Glycopeptide 1.
Assuntos
Anticorpos Monoclonais/imunologia , Distroglicanas/imunologia , Glicoproteínas/imunologia , Mucinas/imunologia , Síndrome de Walker-Warburg/diagnóstico , Distroglicanas/química , Glicoproteínas/síntese química , Humanos , Mucinas/químicaRESUMO
The synthesis of the O-3 triazole-linked galactosyl arylsulfonamides 1-7 as potential inhibitors of Trypanosoma cruzi cell invasion is described. These target compounds were synthesized by Cu(I)-catalysed azide-alkyne cycloaddition reaction ('click chemistry') between different azide arylsulfonamides and the alkyne-based sugar 3-O-propynyl-ßGalOMe. Inhibition assays of T. cruzi cell invasion with compounds 1-7 showed reduced values of infection index (â¼20) for compounds 3 and 5, bearing the corresponding 5-methylisoxazole and 2,4-dimethoxypyrimidine groups, which also presented higher binding affinities to galectin-3 (EC50 17-18⯵M) in Corning Epic label-free assays. In agreement with experimental results, the assessment of the theoretical binding of compounds 1-7 to galectin-3 by MM/PBSA method displayed higher affinities for compounds 3 (-9.7â¯kcal/mol) and 5 (-11.1â¯kcal/mol). Overall, these achievements highlight compounds 3 and 5 as potential T. cruzi cell invasion blockers by means of a galectin-3 binding-related mechanism, revealing galectin-3 as an important host target for design of novel anti-trypanosomal agents.
Assuntos
Inibidores Enzimáticos/farmacologia , Fibroblastos/efeitos dos fármacos , Galectina 3/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Sítios de Ligação/efeitos dos fármacos , Proteínas Sanguíneas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Fibroblastos/parasitologia , Galactose/química , Galactose/farmacologia , Galectinas , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Haplorrinos , Humanos , Estrutura Molecular , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologia , Triazóis/química , Triazóis/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/química , Trypanosoma cruzi/enzimologiaRESUMO
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.