RESUMO
The biological study of O-linked glycosylation is particularly problematic, as chemical tools to control this modification are lacking. An inhibitor of the UDP-GlcNAc 4-epimerase that synthesizes UDP-GalNAc, the donor initiating O-linked glycosylation, would be a powerful reagent for reversibly inhibiting O-linked glycosylation. We synthesized a 1338 member library of uridine analogs directed to the epimerase by virtue of substrate mimicry. Screening of the library identified an inhibitor with a K(i) value of 11 microM. Tests against related enzymes confirmed the compound's specificity for the UDP-GlcNAc 4-epimerase. Inhibitors of a key step of O-linked glycan biosynthesis can be discovered from a directed library screen. Progeny thereof may be powerful tools for controlling O-linked glycosylation in cells.
Assuntos
Carboidratos Epimerases/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Carboidratos Epimerases/metabolismo , Inibidores Enzimáticos/metabolismo , Glicosilação , Humanos , Biblioteca de Peptídeos , Peptídeos/síntese química , Peptídeos/química , Peptídeos/farmacologia , Uridina/metabolismoRESUMO
The polypeptide N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs, also abbreviated ppGaNTases) initiate mucin-type O-linked glycosylation and therefore play pivotal roles in cell-cell communication and protection of tissues. In order to develop new tools for studying mucin-type O-linked glycosylation, we screened a 1338 member uridine-based library to identify small molecule inhibitors of ppGalNAcTs. Using a high-throughput enzyme-linked lectin assay (ELLA), two inhibitors of murine ppGalNAcT-1 (K(I) approximately 8 microM) were identified that also inhibit several other members of the family. The compounds did not inhibit other mammalian glycosyltransferases or nucleotide sugar utilizing enzymes, suggesting selectivity for the ppGalNAcTs. Treatment of cells with the compounds abrogated mucin-type O-linked glycosylation but not N-linked glycosylation and also induced apoptosis. These uridine analogs represent the first generation of chemical tools to study the functions of mucin-type O-linked glycosylation.
Assuntos
Avaliação Pré-Clínica de Medicamentos , Mucinas/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Uridina/análogos & derivados , Uridina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Doxorrubicina/farmacologia , Enzimas Imobilizadas , Glicosilação/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Cinética , Camundongos , Microscopia de Fluorescência , Estrutura Molecular , Mucinas/química , N-Acetilgalactosaminiltransferases/antagonistas & inibidores , N-Acetilgalactosaminiltransferases/metabolismo , Uridina/química , Uridina/isolamento & purificaçãoRESUMO
A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.