RESUMO
We describe the development of cell-permeable beta-secretase inhibitors that demonstratively inhibit the production of the secreted amino terminal fragment of an artificial amyloid precursor protein in cell culture. In addition to potent inhibition in a cell-based assay (IC50 < 100 nM), these inhibitors display impressive selectivity against other biologically relevant aspartyl proteases.
Assuntos
Ácido Aspártico Endopeptidases/antagonistas & inibidores , Etilaminas/síntese química , Sulfonamidas/síntese química , Secretases da Proteína Precursora do Amiloide , Ácido Aspártico Endopeptidases/química , Sítios de Ligação , Linhagem Celular , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Desenho de Fármacos , Etilaminas/química , Etilaminas/farmacologia , Humanos , Modelos Moleculares , Relação Estrutura-Atividade , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
Traditional phenotypic assays used to assess the susceptibility of mutant human immunodeficiency virus type-1 (HIV-1) obtained from infected patients or from resistance selection to antiviral agents in cell culture are rather tedious and time consuming. To improve the efficiency of this process, a novel method was developed in which mutant viruses are captured with magnetic nano-beads and used to infect gag-GFP reporter cells to evaluate the extent of resistance conferred by the mutant viruses against antiviral agents. The optimal timing for measuring the inhibitory potencies of antiviral agents was found to be day 3 post-infection for integrase strand transfer inhibitors and protease inhibitors and day 4 for non-nucleoside reverse transcriptase inhibitors. Comparable EC(50) values were obtained when bead-captured breakthrough virus from in vitro resistance selection experiments and its matched site-directed mutagenesis virus were tested side by side in this assay. This assay protocol was also employed to evaluate the inhibitor susceptibility of breakthrough viruses collected from resistance selections that were conducted in the presence of increasing concentrations of an HIV-1 protease inhibitor. Taken together, these findings suggest that a rapid, sensitive, non-invasive, and homogeneous phenotypic assay has been developed for assessing the antiviral agent susceptibility of mutant viruses that emerge from in vitro resistance selection studies.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Testes de Sensibilidade Microbiana/métodos , Bioensaio , Linhagem Celular , Farmacorresistência Viral Múltipla/genética , Farmacorresistência Viral/genética , Proteínas de Fluorescência Verde/genética , HIV-1/genética , Humanos , Microesferas , Mutagênese Sítio-Dirigida , Mutação , Transfecção , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genéticaRESUMO
Several simple scoring methods were examined for 2 series of beta-secretase (BACE-1) inhibitors to identify a docking/scoring protocol which could be used to design BACE-1 inhibitors in a drug discovery program. Both the PLP1 score and MMFFs interaction energy (E(inter)) performed as well or better than more computationally intensive methods for a set of substrate-based inhibitors, while the latter performed well for both sets of inhibitors.
Assuntos
Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Cristalografia por Raios X , Cinética , Modelos Moleculares , Conformação MolecularRESUMO
A series of beta-site amyloid precursor protein cleaving enzyme (BACE-1) inhibitors containing a psi(CH2NH) reduced amide bond were synthesized. Incorporation of this reduced amide isostere as a non-cleavable peptide surrogate afforded inhibitors possessing low nanomolar potencies in both an enzymatic and cell-based assay.