RESUMO
The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all ß-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of ß-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to ß-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with ß-lactams by preventing the signal peptidase-mediated secretion of proteins required for ß-lactam resistance. Combinations of SpsB inhibitors and ß-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to ß-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.
Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Compostos de Bifenilo/farmacologia , Depsipeptídeos/farmacologia , Glicopeptídeos/farmacologia , Glicosídeos/farmacologia , Lipopeptídeos/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Oligopeptídeos/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , beta-Lactamas/farmacologia , Animais , Antibacterianos/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Transporte Biológico , Compostos de Bifenilo/síntese química , Depsipeptídeos/isolamento & purificação , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Glicopeptídeos/síntese química , Glicopeptídeos/isolamento & purificação , Glicosídeos/isolamento & purificação , Humanos , Lipopeptídeos/isolamento & purificação , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Staphylococcus aureus Resistente à Meticilina/genética , Staphylococcus aureus Resistente à Meticilina/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Família Multigênica , Oligopeptídeos/síntese química , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Infecções Estafilocócicas/microbiologia , Resistência beta-Lactâmica/efeitos dos fármacos , Resistência beta-Lactâmica/genética , beta-Lactamases/genética , beta-Lactamases/metabolismoRESUMO
Activity assays for tyrosine phosphatases are based on the hydrolysis of a arylphosphate moiety from a synthetic substrate yielding a spectroscopically active product. Many different substrates can be used for these assays with p-nitrophenyl phosphate (pNPP), fluorescein diphosphate (FDP), and 6,8-difluoro-4-methylumbellyferyl phosphate (DiFMUP) being the most efficient and versatile. Equally, larger molecules such as phosphotyrosyl peptides can also be used to mimic more natural substrates. Activity assays include the determinations of the rate of dephosphorylation and calculations of kinetic constants such as k(cat) and K(M). These assays are useful to identify and characterize tyrosine phosphatases and are commonly used to evaluate the efficiency of inhibitors.