RESUMEN

Disulfide bond formation has a central role in protein folding of both eukaryotes and prokaryotes. In bacteria, disulfide bonds are catalyzed by DsbA and DsbB/VKOR enzymes. First, DsbA, a periplasmic disulfide oxidoreductase, introduces disulfide bonds into substrate proteins. Then, the membrane enzyme, either DsbB or VKOR, regenerate DsbA's activity by the formation of de novo disulfide bonds which reduce quinone. We have previously performed a high-throughput chemical screen and identified a family of warfarin analogs that target either bacterial DsbB or VKOR. In this work, we expressed functional human VKORc1 in Escherichia coli and performed a structure-activity-relationship analysis to study drug selectivity between bacterial and mammalian enzymes. We found that human VKORc1 can function in E. coli by removing two positive residues, allowing the search for novel anticoagulants using bacteria. We also found one warfarin analog capable of inhibiting both bacterial DsbB and VKOR and a second one antagonized only the mammalian enzymes when expressed in E. coli. The difference in the warfarin structure suggests that substituents at positions three and six in the coumarin ring can provide selectivity between the bacterial and mammalian enzymes. Finally, we identified the two amino acid residues responsible for drug binding. One of these is also essential for de novo disulfide bond formation in both DsbB and VKOR enzymes. Our studies highlight a conserved role of this residue in de novo disulfide-generating enzymes and enable the design of novel anticoagulants or antibacterials using coumarin as a scaffold.

Asunto(s)

Proteínas Bacterianas , Proteínas de Escherichia coli , Escherichia coli , Vitamina K Epóxido Reductasas , Warfarina , Warfarina/metabolismo , Warfarina/química , Vitamina K Epóxido Reductasas/metabolismo , Vitamina K Epóxido Reductasas/química , Vitamina K Epóxido Reductasas/genética , Humanos , Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/enzimología , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Disulfuros/química , Disulfuros/metabolismo , Cumarinas/metabolismo , Cumarinas/química , Proteína Disulfuro Isomerasas/metabolismo , Proteína Disulfuro Isomerasas/química , Proteína Disulfuro Isomerasas/genética , Anticoagulantes/química , Anticoagulantes/metabolismo , Benzoquinonas/metabolismo , Benzoquinonas/química , Relación Estructura-Actividad , Unión Proteica , Proteínas de la Membrana

RESUMEN

Aim: A bacterial genetics-guided approach was utilized for the discovery of new compounds affecting bacterial genome stability. Materials & methods: Fungal extracts and fractions were tested for genome instability-mediated antibacterial activity. Interaction assays and RT-qPCR were used to identify compounds that boost the activity of sub-minimum inhibitory concentration streptomycin and obtain insights on the molecular mechanisms of the primary hit compound, respectively. Results: Several extracts and fractions caused bacterial genome instability. Codeine, in synergy with streptomycin, regulates double-strand break (DSB) repair and causes bacterial ribosome dysfunction in the absence of DSBs, and dysregulation of ribosome biogenesis in a DSB-dependent manner. Conclusion: This study demonstrates a potential viable strategy that we are exploring for the discovery of new chemical entities with activities against Escherichia coli and other bacterial pathogens.