RESUMO
Selective lowering of amyloid-beta levels with small-molecule gamma-secretase inhibitors is a promising therapeutic approach for Alzheimer's disease. In this work, we developed a high throughput assay for screening of gamma-secretase inhibitors with endogenous gamma-secretase and a fluorogenic substrate. The IC(50) values of known gamma-secretase inhibitors generated with this method were comparable with reported values obtained by other methods. The assay was optimized and applied to a small-scale screening of 1,280 compounds. The discovery of several new inhibitors warrants further investigation. This assay was also proven to be easily adopted to test compounds for drosophila and mouse gamma-secretase, which could be very useful to assess compounds activity against gamma-secretase from different species before the in vivo test in animal models.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Drosophila melanogaster/enzimologia , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/análise , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Sequência de Aminoácidos , Secretases da Proteína Precursora do Amiloide/química , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Camundongos , Dados de Sequência MolecularRESUMO
OBJECTIVE: To clone and express the recombinant amyloid beta-protein (Abeta(42)) antigen and develop a method for detecting Abeta antibody. METHODS: Two partially complementary fragments of Abeta(42) gene were chemically synthesized for constructing the Abeta(42) gene by PCR. The resultant Abeta(42) gene fragment was subcloned into pGEX-2T expression vector for inducing the expression of GST-Abeta(42) fusion protein, which was purified by affinity chromatography. The antigen specificity and reactivity of the purified GST-Abeta(42) fusion protein to Abeta monoclonal antibody were identified with Western blotting. Using either GST-Abeta(42) fusion protein or Abeta(42) peptide as the coating antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was established for detecting Abeta antibodies in the serum of Abeta(42) polypeptide-immunized SD rats. RESULTS: GST-Abeta(42) fusion protein was successfully expressed as an soluble protein, which, after purification, was found to have a relative molecular mass of 31 kD. About 800 mg of GST-Abeta(42) fusion protein were obtained from l L cell culture with a purity over 95%. Western blotting demonstrated specific reaction of this purified GST-Abeta(42) fusion protein with Abeta monoclonal antibody. The sensitivity of the indirect ELISA for detecting Abeta antibody was about 2 ng/ml using GST-Abeta(42) fusion protein as the coating antigen. There was no significant difference in the results of Abeta antibody detection using either GST-Abeta(42) or Abeta(42) as the coating antigen (P>0.05). CONCLUSION: GST-Abeta(42) fusion protein may serve as a substitute for the expensive Abeta(42) peptide for detecting Abeta antibody.
