INVESTIGADORES
VILCHEZ LARREA Salome Catalina
congresos y reuniones científicas
Título:
In vitro characterization and inhibition of Trypanosoma cruzi and brucei PARPs
Autor/es:
SALOMÉ VILCHEZ LARREA; MARIANA SCHLESINGER; MOHIT NARWAL; SILVIA H. FERNÁNDEZ VILLAMIL; LARI LEHTIÖ
Lugar:
Zurich, Suiza
Reunión:
Conferencia; 18th International Conference on ADP-ribose metabolism; 2010
Resumen:
Trypanosoma cruzi and Trypanosoma brucei are protozoan parasites causing major public health problems in South America and Africa, respectively. In contrast to humans, these trypanosomatids have only one PARP enzyme (TcPARP or TbPARP), which in vivo is activated by DNA damage (1). We have expressed both PARPs in bacterial systems and purified recombinant proteins for further activity analysis. According to size exclusion chromatography, the most active form of both recombinant TcPARP and TbPARP is a dimer. We optimized assay conditions for both enzymes, which share similar properties regarding their activity requirements e.g. optimal pH being 7. Despite the fact that none of them show characterized DNA binding domains, both enzymes are activated in vitro by nicked DNA. Notably, neither of them requires Mg2+ or other metal ions. Instead, as we previously showed for TcPARP (1), PARPs from these trypanosomatids are inhibited by all the other divalent cations tested. A small panel of compounds consisting mostly of known PARP inhibitors was tested for inhibitory capacity on TcPARP and TbPARP. Compounds show very similar inhibition of both enzymes, while many of the inhibitors tested did not inhibit either significantly at 10 µM concentration. The most potent inhibitors tested were Olaparib and EB-47, the latter showing 10-fold selectivity for trypanosome enzymes over human PARP-1. These inhibitors among others will be next tested for their ability to affect parasites PARP activity in vivo, as well as the effects of this inhibition on the parasites DNA damage response, post-stress survival and cell cycle.