IN VITRO CHARACTERIZATION AND INHIBITION OF Trypanosoma cruzi AND Trypanosoma brucei PARP

VILCHEZ LARREA S C; SCHLESINGER M; MOHIT NARWAL; TORRES H N; FLAWIA M M; LARI LEHTIÖ; FERNANDEZ VILLAMIL S H

Puerto Madryn

Congreso; 46 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2010

SAIB

In contrast to humans, Trypanosoma cruzi and T. brucei, causal agents of American and African trypanosomosis respectively, have only one PARP enzyme (TcPARP or TbPARP), which in vivo is activated by DNA damage. 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. 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 previously shown for TcPARP, trypanosomatid PARPs are inhibited by many divalent cations. A panel of compounds known as PARP inhibitors was tested for inhibitory capacity on TcPARP and TbPARP. While some compounds did not inhibit either significantly at 10 μM concentration, most showed very similar inhibition of both enzymes. The most potent inhibitors tested were Olaparib and EB-47, the latter showing 10-fold selectivity for trypanosome enzymes over hPARP-1. Inhibitors will be tested for their ability to affect parasites PARP activity in vivo, as well as its effects on DNA damage response, post-stress survival and cell cycle.