%0 Generic
%A García Esteban, María Teresa
%A Blázquez, M.A.
%A Ferrándiz, M.J.
%A Sanz, M.J.
%A Martín, N.S.
%A Hermoso, J.A.
%A Campa, A.G. de la
%T New Boldine-derived antibiotics against DNA Topoisomerase I of Streptococcus pneumoniae
%D 2010
%U https://hdl.handle.net/20.500.14352/130352
%X Background: DNA topoisomerases are essential enzymes that control DNA topology. Thetopoisomerase complement of Streptococcus pneumoniae consists of 2 type 11 enzymes(gyrase and topoisomerase IV) and a unique type 1 enzyme (topoisomerase 1). Whilefluoroquinolone antibiotics target the type 11 enzymes, no antibiotic that targets efficientlytopoisomerase 1 (TopA} has yet been described.Methods: 18 synthetic alkaloids were used to test inhibition of growth of multiresistantpneumococcal isolates and of pneumococcal TopA activity. Bidimensional agarose gelelectrophoresis containing chloroquine was used to study supercoiling levels. Molecularmodelling of pneumococcal TopA was built on the basis of the crystal structure of his E. colihomolog. Docking analysis of TopA with alkaloids was carried out.Results: The pneumococcal topA gene was cloned in E. coli and overexpressed as a fusionprotein with an N-terminal-6His tag. TopA showed nicking-closing activity on negativelysupercoiledplasmid DNA. This activity, as well as cell growth, was inhibited in vitro with 17 μMof seconeolitsine and N-methly-seconeolitsine. However, the inhibitory effect of seconeolitsinedecreased when TopA was overproduced. This protection, and the hypernegative supercoilingexhibited by an interna! plasmid after treatment with the alkaloid, support the in vivo targeting ofTopA. This inhibition might be dueto strong interactions between the alkaloids and thenucleotide-binding site in the protein's closed conformation that should block the openingmechanism required for topo 1 to bind DNA.Conclusions: TopA is the intracellular target of 2 synthetic phenanthrene alkaloids that couldbe used as new antibiotics for the treatment of diseases caused by multiresistant isolates.
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