The essential and major biosynthetic step in the me tabolism of all isoprenoid is the elongation of isoprene units by prenyltransferases. These enzymes, which subsequently mediate alkylation of IPP by allylic di phosphate, are classified according to the chain length of the final product and stereochemistry U0126 MEK inhibitor of the double bond formed by condensations. FPPS and GGPPS are the most studied prenyltransferases and have been de scribed in various organisms of all three kingdoms, Eukarya, Bacteria, and Archaea. In protist para sites, the FPPS gene was cloned from Trypanosoma cruzi, Trypanosoma brucei and Toxoplasma gondii. Recently, a GGPPS from Plasmodium vivax was described. However, the first characterization of a prenyltransferase in a malaria parasite was the characterization of the octaprenyl diphosphate synthase that catalyzes the condensation of FPP with IPP to produce octaprenyl diphosphate.

Human FPPS has Inhibitors,Modulators,Libraries been found to be a target for nitrogen containing bisphosphonate drugs. Based on growth rescue and enzyme inhibition experi ments, human GGPPS was shown to be a major target for the lipophilic analogues zolendronate and risedronate. These reports have generated considerable interest in FPPS as a promising target for new anti malarial drug development. Jord?o et al. suggested the possible mechanism of action for risedronate in P. falciparum by inhibition of FPPS. In the causative agent of sleeping sickness, T. brucei, the inhibition of FPPS showed that Inhibitors,Modulators,Libraries this enzyme is essential for parasite sur vival.

Considering that FPPS is a key enzyme of the biosynthesis of compounds already characterized in the parasite, such as dolichols, farnesylated pro teins, and other final isoprenoid products, it is es sential to characterize the FPPS from P. falciparum in order to establish an appropriate strategy Inhibitors,Modulators,Libraries for Inhibitors,Modulators,Libraries the de velopment of specific inhibitors. This work describes the cloning, expression and characterization of recombinant P. falciparum FPPS, with catalytic activity for DMAPP, GPP, and FPP as substrates, yielding FPP and GGPP as final prod ucts. Apparent kinetic parameters for the recombinant enzyme are presented, as well as IC50 and apparent Ki values for risedronate inhibition of rPfFPPS enzyme ac tivity. Constitutive protein expression is also described. Methods Plasmodium falciparum culture Cultures of P.

falciparum clone 3D7 were grown as described, replacing human serum with Albumax I. Parasite development and multiplication were monitored by microscopic evaluation of Giemsa stained thin smears. Schizont stages were purified with magnetic columns. Inhibitors,Modulators,Libraries Column pre equilibration, washing and elution were all carried out at room temperature with RPMI 1640. selleck inhibitor For schizont purification, the culture was centrifuged, the pel let resuspended in RMPI 1640, 10 ml of the 10% suspension of erythrocytes were applied to a CS column assembled in a magnetic unit, where only schizonts are retained.