New Boldine-derived antibiotics against DNA Topoisomerase I of Streptococcus pneumoniae

Abstract

Background: DNA topoisomerases are essential enzymes that control DNA topology. The topoisomerase complement of Streptococcus pneumoniae consists of 2 type 11 enzymes (gyrase and topoisomerase IV) and a unique type 1 enzyme (topoisomerase 1). While fluoroquinolone antibiotics target the type 11 enzymes, no antibiotic that targets efficiently topoisomerase 1 (TopA} has yet been described. Methods: 18 synthetic alkaloids were used to test inhibition of growth of multiresistant pneumococcal isolates and of pneumococcal TopA activity. Bidimensional agarose gel electrophoresis containing chloroquine was used to study supercoiling levels. Molecular modelling of pneumococcal TopA was built on the basis of the crystal structure of his E. coli homolog. Docking analysis of TopA with alkaloids was carried out. Results: The pneumococcal topA gene was cloned in E. coli and overexpressed as a fusion protein with an N-terminal-6His tag. TopA showed nicking-closing activity on negativelysupercoiled plasmid DNA. This activity, as well as cell growth, was inhibited in vitro with 17 μM of seconeolitsine and N-methly-seconeolitsine. However, the inhibitory effect of seconeolitsine decreased when TopA was overproduced. This protection, and the hypernegative supercoiling exhibited by an interna! plasmid after treatment with the alkaloid, support the in vivo targeting of TopA. This inhibition might be dueto strong interactions between the alkaloids and the nucleotide-binding site in the protein's closed conformation that should block the opening mechanism required for topo 1 to bind DNA. Conclusions: TopA is the intracellular target of 2 synthetic phenanthrene alkaloids that could be used as new antibiotics for the treatment of diseases caused by multiresistant isolates.