RESUMO
Natural products harbor unique and complex structures that provide valuable antibiotic scaffolds. With an increase in antibiotic resistance, natural products once again hold promise for new antimicrobial therapies, especially those with unique scaffolds that have been overlooked due to a lack of understanding of how they function. Dithiolopyrrolones (DTPs) are an underexplored class of disulfide-containing natural products, which exhibit potent antimicrobial activities against multidrug-resistant pathogens. DTPs were thought to target RNA polymerase, but conflicting observations leave the mechanisms elusive. Using a chemical genomics screen in Escherichia coli, we uncover a mode of action for DTPs-the disruption of metal homeostasis. We show that holomycin, a prototypical DTP, is reductively activated, and reduced holomycin chelates zinc with high affinity. Examination of reduced holomycin against zinc-dependent metalloenzymes revealed that it inhibits E. coli class II fructose bisphosphate aldolase, but not RNA polymerase. Reduced holomycin also strongly inhibits metallo-ß-lactamases in vitro, major contributors to clinical carbapenem resistance, by removing active site zinc. These results indicate that holomycin is an intracellular metal-chelating antibiotic that inhibits a subset of metalloenzymes and that RNA polymerase is unlikely to be the primary target. Our work establishes a link between the chemical structures of DTPs and their antimicrobial action; the ene-dithiol group of DTPs enables high-affinity metal binding as a central mechanism to inhibit metabolic processes. Our study also validates the use of chemical genomics in characterizing modes of actions of antibiotics and emphasizes the potential of metal-chelating natural products in antimicrobial therapy.
Assuntos
Escherichia coli/efeitos dos fármacos , Lactamas/farmacologia , Pirróis/química , Tolueno/análogos & derivados , Antibacterianos/química , Antibacterianos/uso terapêutico , Produtos Biológicos/química , Produtos Biológicos/uso terapêutico , Domínio Catalítico/efeitos dos fármacos , RNA Polimerases Dirigidas por DNA/antagonistas & inibidores , RNA Polimerases Dirigidas por DNA/genética , Dissulfetos/química , Dissulfetos/uso terapêutico , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Genômica , Homeostase/efeitos dos fármacos , Humanos , Lactamas/química , Metaloproteínas/antagonistas & inibidores , Metaloproteínas/genética , Metais/química , Pirróis/uso terapêutico , Tolueno/química , Tolueno/uso terapêutico , Zinco/metabolismo , beta-Lactamases/efeitos dos fármacos , beta-Lactamases/genéticaRESUMO
A panel of iron (Fe) and copper (Cu) chelators was screened for growth inhibitory activity against the fungal pathogen Cryptococcus neoformans. Select bidentate metal-binding ligands containing mixed O,S or O,N donor atoms were identified as agents that induce cell killing in a Cu-dependent manner. Conversely, structurally similar ligands with O,O donor atoms did not inhibit C. neoformans growth regardless of Cu status. Studies of Cu(ii) and Cu(i) binding affinity, lipophilicity, and growth recovery assays of Cu-import deficient cells identified lipophilicity of thermodynamically stable CuIIL2 complexes as the best predictor of antifungal activity. These same complexes induce cellular hyperaccumulation of Zn and Fe in addition to Cu. The results described here present the utility of appropriate metal-binding ligands as potential antifungal agents that manipulate cellular metal balance as an antimicrobial strategy.