RESUMO
We report the results of an in vitro screening assay targeting the intraerythrocytic form of the malaria parasite Plasmodium falciparum using a library of 560 prenyl-synthase inhibitors. Based on "growth-rescue" and enzyme-inhibition experiments, geranylgeranyl diphosphate synthase (GGPPS) is shown to be a major target for the most potent leads, BPH-703 and BPH-811, lipophilic analogs of the bone-resorption drugs zoledronate and risedronate. We determined the crystal structures of these inhibitors bound to a Plasmodium GGPPS finding that their head groups bind to the [Mg(2+)](3) cluster in the active site in a similar manner to that found with their more hydrophilic parents, whereas their hydrophobic tails occupy a long-hydrophobic tunnel spanning both molecules in the dimer. The results of isothermal-titration-calorimetric experiments show that both lipophilic bisphosphonates bind to GGPPS with, on average, a ΔG of -9 kcal mol(-1), only 0.5 kcal mol(-1) worse than the parent bisphosphonates, consistent with the observation that conversion to the lipophilic species has only a minor effect on enzyme activity. However, only the lipophilic species are active in cells. We also tested both compounds in mice, finding major decreases in parasitemia and 100% survival. These results are of broad general interest because they indicate that it may be possible to overcome barriers to cell penetration of existing bisphosphonate drugs in this and other systems by simple covalent modification to form lipophilic analogs that retain their enzyme-inhibition activity and are also effective in vitro and in vivo.
Assuntos
Antimaláricos/farmacologia , Difosfonatos/farmacologia , Ácido Etidrônico/análogos & derivados , Farnesiltranstransferase/antagonistas & inibidores , Imidazóis/farmacologia , Lipídeos/química , Plasmodium/efeitos dos fármacos , Plasmodium/enzimologia , Animais , Antimaláricos/química , Antimaláricos/uso terapêutico , Calorimetria , Cristalografia por Raios X , Difosfonatos/química , Difosfonatos/uso terapêutico , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Eritrócitos/efeitos dos fármacos , Eritrócitos/parasitologia , Ácido Etidrônico/química , Ácido Etidrônico/farmacologia , Ácido Etidrônico/uso terapêutico , Farnesiltranstransferase/química , Farnesiltranstransferase/metabolismo , Ensaios de Triagem em Larga Escala , Humanos , Imidazóis/química , Imidazóis/uso terapêutico , Camundongos , Modelos Moleculares , Parasitemia/tratamento farmacológico , Parasitemia/parasitologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Plasmodium vivax/efeitos dos fármacos , Plasmodium vivax/enzimologia , Ligação Proteica/efeitos dos fármacos , Ácido Risedrônico , Análise de Sobrevida , Terpenos/química , Terpenos/metabolismo , Ácido ZoledrônicoRESUMO
The protozoan parasite Trypanosoma cruzi is the etiologic agent of Chagas disease, a neglected tropical infection that affects millions of people in the Americas. Current chemotherapy relies on only two drugs that have limited efficacy and considerable side effects. Therefore, the development of new and more effective drugs is of paramount importance. Although some host cellular factors that play a role in T. cruzi infection have been uncovered, the molecular requirements for intracellular parasite growth and persistence are still not well understood. To further study these host-parasite interactions and identify human host factors required for T. cruzi infection, we performed a genome-wide RNAi screen using cellular microarrays of a printed siRNA library that spanned the whole human genome. The screening was reproduced 6 times and a customized algorithm was used to select as hits those genes whose silencing visually impaired parasite infection. The 162 strongest hits were subjected to a secondary screening and subsequently validated in two different cell lines. Among the fourteen hits confirmed, we recognized some cellular membrane proteins that might function as cell receptors for parasite entry and others that may be related to calcium release triggered by parasites during cell invasion. In addition, two of the hits are related to the TGF-beta signaling pathway, whose inhibition is already known to diminish levels of T. cruzi infection. This study represents a significant step toward unveiling the key molecular requirements for host cell invasion and revealing new potential targets for antiparasitic therapy.