Isoprenoids are an extensive group of natural products with diverse structures consisting of various numbers of five carbon isopentenyl pyrophosphate (IPP) units. The isoprenoid pathway has been studied in mammalian cells where the enzyme farnesyl pyrophosphate synthase (FPPS) plays a central role by producing FPP, an important precursor of sterols, dolichols, ubiquinones, and prenylated proteins. FPP, a 15 C isoprenoid unit could further form GGPP, a 15 C isoprenoid unit by the enzyme geranylgeranyl pyrophosphate synthase (GGPPS). FPPS forms FPP by the sequential condensation of dimethylallylpyrophosphate (DMAPP) with two molecules of isopentenylpyrophosphate (IPP). Little is known about the isoprenoid pathway in Toxoplasma gondii. Taking into account the central role of FPPS, its understanding will reveal important information on this pathway in T. gondii. In addition, our preliminary results indicate that this enzyme is a valid target for drugs since bisphosphonates, which are specific FPPS inhibitors, inhibited parasite growth in vitro and in vivo. The gene for the T. gondii farnesyl pyrophosphate synthase (TgFFPS) was cloned, sequenced and characterized. This gene was present as a single copy in the tachyzoite haploid genome, although by RT-PCR two transcripts were found and named TgFPPSI and TgFPPSII. Both isoforms of TgFPPS were heterologously expressed in the Bac-to-Bac expression systems and the corresponding protein(s) purified for their biochemical characterization. Only isoform II had enzymatic activity and, interestingly, this enzyme was capable of forming both products, farnesyl pyrophosphate and geranyl geranyl pyrophosphate. This unique characteristic was previously found only in enzymes of the Archaea. Another interesting finding was the mitochondrial localization of this enzyme, which agrees with the presence of a mitochondrial-targeting signal in the protein sequence. This is also a unique feature of this protein since other FPPS are normally found in the cytosol. It has been demonstrated that the FPPS is the target for bisphosphonates, FDA-approved pyrophosphate analogs currently used in the treatment of bone resorption disorders. These compounds inhibited the proliferation of T. gondii in vitro and in vivo. Our preliminary results show that there was a significant difference between the sensitivity of the TgFPPS enzyme to bisphosphonates when compared with the human counterpart. Our hypothesis is that the isoprenoid pathway constitutes a major novel target for the treatment of toxoplasmosis. To test this hypothesis, our specific aims are:(1) To further investigate the TgFPPS by site-directed mutagenesis to understand its bifunctionality, to perform structural studies to facilitate drug design, and to investigate the importance of TgFPPS products in the parasite biology; (2) To investigate the effect of pyrophosphate analogs in vitro against T. gondii, and perform structure-activity relationship studies to facilitate drug design. ? ? ?
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