Proceedings book download - 5Z.com

The selected peptides were assayed for determination of the minimum concentration thatproduced complete inhibition of supercoiling and relaxation activities (termed IC 100 ). Instandard supercoiling assays at 37°C with 3.4 nmol.L -1 of gyrase, a relaxed DNA (500 ng)substrate is completely negatively supercoiled in 1h [4]. EcParE3, EcParE8 and EcParE10inhibited this reaction (Figure 3A) with IC 100 values of 25, 50 and 25 µmol.L -1 respectively.Fig. 3. EcParE peptides-mediated inhibition of (A) DNA supercoiling reactions of DNAgyrase and (B) DNA relaxation reactions of Topo IV. Controls: C1: negative (absence ofpeptide and enzyme); C2: positive (absence of peptide); The numbers in the lanesrepresents the concentration of the peptides in µmol.L -1 .In addition, EcParE3, EcParE8 and EcParE10 inhibited the ATP-dependent relaxationreaction of Topo IV, in standard assays (37°C, 5 nmol.L -1 of enzyme and 400 ng ofsupercoiled DNA) with IC 100 values of 5, 25 and 10 µmol.L -1 (Figure 3B). For both, there isno evidence of cleavage complex. From these results it is possible to speculate about of therequirement of the C-terminal LNIES sequence in a probable structure-activity relationshipof the peptides synthesized. Comparatively EcParE3 and EcParE10, the best inhibitors ofboth Gyrase and Topo IV, do not include this sequence in its primary structure. Probablypeptides with this sequence do not have the adequate molecular adjustments for theformation of an inactive complex with the enzymes. Our preliminary findings revealed anew class of peptide inhibitors of bacterial topoisomerases and suggest that DNA gyrase aswell as Topo IV may be effective targets of the natural ParE toxin.AcknowledgmentsThis work was supported by FAPESP in the form of grant (10/07841-2) and by CNPq in the Ph.Dfellowship form (L.C.B. Barbosa) R. Marchetto is the recipient of research fellowship from CNPq.References1. Jiang, Y., Pogliano, J., Helinski, D.R., Konieczny, I. Mol. Microbiol. 44, 971-979 (2002).2. Barbosa, L.C.B., Garrido, S.S., Garcia, A., Delfino, D.B., Marchetto, R. Bioinformation 4, 438-440(2010).3. Fiebig, A., Rojas, C.M.C., Siegal-Gaskins, D., Crosson, S. Mol. Microbiol. 77, 236-251 (2010).4. Trovatti, E., Cotrim, C.A., Garrido, S.S., Barros, R., Marchetto, R. Bioorg. Med. Chem. Lett. 18,6161-6164 (2008).483