RESUMO
Antibiotic-resistant strains of Staphylococcus aureus pose a major threat to human health and there is an ongoing need for new antibiotics to treat resistant infections. In a high throughput screen (HTS) of 230â¯000 small molecules designed to identify bioactive wall teichoic acid (WTA) inhibitors, we identified one hit, which was expanded through chemical synthesis into a small panel of potent compounds. We showed that these compounds target TarG, the transmembrane component of the two-component ATP-binding cassette (ABC) transporter TarGH, which exports WTA precursors to the cell surface for attachment to peptidoglycan. We purified, for the first time, a WTA transporter and have reconstituted ATPase activity in proteoliposomes. We showed that this new compound series inhibits TarH-catalyzed ATP hydrolysis even though the binding site maps to TarG near the opposite side of the membrane. These are the first ABC transporter inhibitors shown to block ATPase activity by binding to the transmembrane domain. The compounds have potential as therapeutic agents to treat S. aureus infections, and purification of the transmembrane transporter will enable further development.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Ácidos Teicoicos/farmacologia , Adenosina Trifosfatases/antagonistas & inibidores , Sítios de Ligação , Parede Celular/química , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Modelos Biológicos , Estrutura Molecular , Peptidoglicano/química , Peptidoglicano/metabolismo , Ligação Proteica/efeitos dos fármacosRESUMO
The majority of bacterial proteins are dispensable for growth in the laboratory but nevertheless have important physiological roles. There are no systematic approaches to identify cell-permeable small-molecule inhibitors of these proteins. We demonstrate a strategy to identify such inhibitors that exploits synthetic lethal relationships both for small-molecule discovery and for target identification. Applying this strategy in Staphylococcus aureus, we have identified a compound that inhibits DltB, a component of the teichoic acid D-alanylation machinery that has been implicated in virulence. This D-alanylation inhibitor sensitizes S. aureus to aminoglycosides and cationic peptides and is lethal in combination with a wall teichoic acid inhibitor. We conclude that DltB is a druggable target in the D-alanylation pathway. More broadly, the work described demonstrates a systematic method to identify biologically active inhibitors of major bacterial processes that can be adapted to numerous organisms.