RESUMEN
We report the cloning of a Trypanosoma cruzi gene encoding a solanesyl-diphosphate synthase, TcSPPS. The amino acid sequence (molecular mass approximately 39 kDa) is homologous to polyprenyl-diphosphate synthases from different organisms, showing the seven conserved motifs and the typical hydrophobic profile. TcSPPS preferred geranylgeranyl diphosphate as the allylic substrate. The final product, as determined by TLC, had nine isoprene units. This suggests that the parasite synthesizes mainly ubiquinone-9 (UQ-9), as described for Trypanosoma brucei and Leishmania major. In fact, that was the length of the ubiquinone extracted from epimastigotes, as determined by high-performance liquid chromatography. Expression of TcSPPS was able to complement an Escherichia coli ispB mutant. A punctuated pattern in the cytoplasm of the parasite was detected by immunofluorescence analysis with a specific polyclonal antibody against TcSPPS. An overlapping fluorescence pattern was observed using an antibody directed against the glycosomal marker pyruvate phosphate dikinase, suggesting that this step of the isoprenoid biosynthetic pathway is located in the glycosomes. Co-localization in glycosomes was confirmed by immunogold electron microscopy and subcellular fractionation. Because UQ has a central role in energy production and in reoxidation of reduction equivalents, TcSPPS is promising as a new chemotherapeutic target.
Asunto(s)
Transferasas Alquil y Aril/biosíntesis , Microcuerpos/metabolismo , Trypanosoma cruzi/metabolismo , Transferasas Alquil y Aril/química , Secuencia de Aminoácidos , Animales , Cromatografía en Capa Delgada , Clonación Molecular , Cósmidos , Escherichia coli/metabolismo , Prueba de Complementación Genética , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Ubiquinona/química , Ubiquinona/aislamiento & purificaciónRESUMEN
Studies on the mode of action of a series of bisphosphonates derived from fatty acids, which had previously proved to be potent inhibitors against Trypanosoma cruzi proliferation in in vitro assays, have been performed. Some of these drugs proved to be potent inhibitors against the intracellular form of the parasite, exhibiting IC(50) values at the low micromolar level. As bisphosphonates are FDA clinically approved for treatment of bone resorption disorders, their potential innocuousness makes them good candidates to control tropical diseases.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Difosfonatos/farmacología , Inhibidores Enzimáticos/farmacología , Ácidos Grasos/farmacología , Trypanosoma cruzi/efectos de los fármacos , Animales , Difosfonatos/química , Difosfonatos/aislamiento & purificación , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Ácidos Grasos/química , Ácidos Grasos/aislamiento & purificación , Geraniltranstransferasa , Trypanosoma cruzi/enzimologíaRESUMEN
We investigated the molecular basis of the activity of 4-phenoxyphenoxyethyl thiocyanate (WC-9) against Trypanosoma cruzi, the etiological agent of Chagas' disease. We found that growth inhibition of T. cruzi epimastigotes induced by this compound was associated with a reduction in the content of the parasite's endogenous sterols due to a specific blockade of their de novo synthesis at the level of squalene synthase.