RESUMO
Natural products serve as chemical blueprints for most antibiotics in clinical use. The evolutionary process by which these molecules arise is inherently accompanied by the co-evolution of resistance mechanisms that shorten the clinical lifetime of any given class of antibiotics1. Virginiamycin acetyltransferase (Vat) enzymes are resistance proteins that provide protection against streptogramins2, potent antibiotics against Gram-positive bacteria that inhibit the bacterial ribosome3. Owing to the challenge of selectively modifying the chemically complex, 23-membered macrocyclic scaffold of group A streptogramins, analogues that overcome the resistance conferred by Vat enzymes have not been previously developed2. Here we report the design, synthesis, and antibacterial evaluation of group A streptogramin antibiotics with extensive structural variability. Using cryo-electron microscopy and forcefield-based refinement, we characterize the binding of eight analogues to the bacterial ribosome at high resolution, revealing binding interactions that extend into the peptidyl tRNA-binding site and towards synergistic binders that occupy the nascent peptide exit tunnel. One of these analogues has excellent activity against several streptogramin-resistant strains of Staphylococcus aureus, exhibits decreased rates of acetylation in vitro, and is effective at lowering bacterial load in a mouse model of infection. Our results demonstrate that the combination of rational design and modular chemical synthesis can revitalize classes of antibiotics that are limited by naturally arising resistance mechanisms.
Assuntos
Antibacterianos/síntese química , Antibacterianos/farmacologia , Desenho de Fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Estreptogramina Grupo A/síntese química , Estreptogramina Grupo A/farmacologia , Acetilação/efeitos dos fármacos , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Antibacterianos/classificação , Carga Bacteriana/efeitos dos fármacos , Sítios de Ligação , Microscopia Crioeletrônica , Feminino , Técnicas In Vitro , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , RNA de Transferência/metabolismo , Ribossomos/efeitos dos fármacos , Ribossomos/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Estreptogramina Grupo A/química , Estreptogramina Grupo A/classificação , Virginiamicina/análogos & derivados , Virginiamicina/química , Virginiamicina/metabolismoAssuntos
Transportadores de Cassetes de Ligação de ATP/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana Múltipla/genética , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Plasmídeos/genética , Doenças dos Suínos/microbiologia , Animais , Diterpenos/farmacologia , Feminino , Lincosamidas/farmacologia , Staphylococcus aureus Resistente à Meticilina/genética , Testes de Sensibilidade Microbiana , Compostos Policíclicos , Recombinação Genética , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/veterinária , Estreptogramina Grupo A/farmacologia , Suínos , Zoonoses/microbiologiaRESUMO
Virginiamycin acetyltransferase D (VatD) plays a vital rule in streptogramins resistance by chemically inactivating streptogramin A. Therefore, it is desirable to discover novel small molecular weight inhibitors of VatD via state-of-the-art virtual screening techniques. This "cocktail" strategy by combining VatD inhibitor with streptogramins may provide new therapeutic opportunity for resistant bacteria infections. Structure-based virtual screening method (molecular docking) was applied to rank and score a chemical database containing 300 000 commercially available compounds against the VatD substrate binding site. Twenty six out of the 200 top scored compounds from the docking calculation were selected and submitted to the VatD enzymatic inhibition assay. The plasmid pRSET B/vatD was constructed and transformed into E. coli (trxB) host cells for over-expression, and VatD enzyme was purified and validated by showing acetyltransferase activity to Virginiamycin M1. Three out of these 26 tested compounds showed enzymatic inhibition on VatD with IC50 168.6, 91.0 and 55.2 micromol x L(-1), separately. Other compounds could not be dissolved in the system and/or had little effect on the enzyme (IC50 > 200 micromol x L(-1)). To our knowledge, it is first time that small molecular weight organic compounds were identified as VatD inhibitors. It is expected that the VatD inhibitors identified at present study could serve as lead compounds for the further development of the novel therapeutic agents to overcome streptogramins resistance.
Assuntos
Acetiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Estreptogramina Grupo A/farmacologia , Acetiltransferases/genética , Acetiltransferases/metabolismo , Catálise/efeitos dos fármacos , Desenho de Fármacos , Farmacorresistência Bacteriana , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Escherichia coli/genética , Vetores Genéticos , Cinética , Estrutura Molecular , Plasmídeos , Estreptogramina Grupo A/química , Estreptogramina Grupo A/metabolismo , Transformação GenéticaRESUMO
The streptogramin antibiotics were discovered over 40 years ago but are only now emerging as important therapeutic agents for the treatment of infection caused by a variety of bacteria. The streptogramins consist of mixtures of two structurally distinct compounds, type A and type B, which are separately bacteriostatic, but bactericidal in appropriate ratios. These antibiotics act at the level of inhibition of translation through binding to the bacterial ribosome. Resistance to streptogramins occurs through a number of mechanisms including target modification, efflux, and enzyme catalyzed antibiotic modification. This review describes the current understanding of streptogramin function and resistance with emphasis on molecular mechanism and epidemiology.
