RESUMO
The Salmonella PmrA/PmrB two-component system uses an iron(III)-binding motif on the cell surface to sense the environmental or host ferric level and regulate PmrA-controlled gene expression. We replaced the iron(III)-binding motif with a lanthanide-binding peptide sequence that is known to selectively recognize trivalent lanthanide ions. The newly engineered two-component system (PmrA/PmrB) can effectively sense lanthanide ion and regulate gene expression in E. coli . This work not only provides the first known lanthanide-based sensing and response in live cells but also demonstrates that the PmrA/PmrB system is a suitable template for future synthetic biology efforts to construct bacteria that can sense and respond to other metal ions in remediation or sequestration.
Assuntos
Proteínas de Bactérias/metabolismo , Elementos da Série dos Lantanídeos/análise , Engenharia de Proteínas , Salmonella typhimurium , Fatores de Transcrição/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Compostos Férricos/química , Compostos Férricos/metabolismo , Íons/análise , Íons/metabolismo , Elementos da Série dos Lantanídeos/metabolismo , Modelos Biológicos , Peptídeos/química , Peptídeos/metabolismo , Salmonella typhimurium/genética , Fatores de Transcrição/química , Fatores de Transcrição/genéticaRESUMO
A series of diketo tetrazoles and diketo triazoles were designed and synthesized as bioisosteres of α,γ-diketo acid, the active site inhibitor of HCV (Hepatitis C virus) polymerase NS5B. Among the synthesized compounds, 4-(4-fluorobenzyloxy)phenyl diketo triazole (30) exhibited anti-HCV activity with an EC50 value of 3.9 µM and an SI value more than 128. The reduction of viral protein and mRNA levels were also validated, supporting the anti-HCV activity of compound 30. These results provide convincing evidence that the diketo tetrazoles and diketo triazoles can be developed as bioisosteres of α,γ-diketo acid to exhibit potent inhibitory activity against HCV.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Ácidos Carboxílicos/síntese química , Ácidos Carboxílicos/farmacologia , Descoberta de Drogas , Hepacivirus/efeitos dos fármacos , Antivirais/química , Ácidos Carboxílicos/química , Relação Dose-Resposta a Droga , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Tetrazóis/síntese química , Tetrazóis/química , Tetrazóis/farmacologia , Triazóis/síntese química , Triazóis/química , Triazóis/farmacologiaRESUMO
Uranyl (UO2(2+)), the predominant aerobic form of uranium, is present in the ocean at a concentration of ~3.2 parts per 10(9) (13.7 nM); however, the successful enrichment of uranyl from this vast resource has been limited by the high concentrations of metal ions of similar size and charge, which makes it difficult to design a binding motif that is selective for uranyl. Here we report the design and rational development of a uranyl-binding protein using a computational screening process in the initial search for potential uranyl-binding sites. The engineered protein is thermally stable and offers very high affinity and selectivity for uranyl with a Kd of 7.4 femtomolar (fM) and >10,000-fold selectivity over other metal ions. We also demonstrated that the uranyl-binding protein can repeatedly sequester 30-60% of the uranyl in synthetic sea water. The chemical strategy employed here may be applied to engineer other selective metal-binding proteins for biotechnology and remediation applications.