Characterization of potential drug targets farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase in Schistosoma mansoni

Abstract

Schistosomiasis affects over 200 million people worldwide, with over 200,000 deaths annually. Currently, praziquantel is the only drug available against schistosomiasis. We report here that Schistosoma mansoni farnesyl diphosphate synthase (SmFPPS) and geranylgeranyl diphosphate synthase (SmGGPPS) are potential drug targets for the treatment of schistosomiasis. We expressed active, recombinant SmFPPS and SmGGPPS for subsequent kinetic characterization and testing against a variety of bisphosphonate inhibitors. Recombinant SmFPPS was found to be a soluble 44.2-kDa protein, while SmGGPPS was a soluble 38.3-kDa protein. Characterization of the substrate utilization of the two enzymes indicates that they have overlapping substrate specificities. Against SmFPPS, several bisphosphonates had 50% inhibitory concentrations (IC50s) in the low micromolar to nanomolar range; these inhibitors had significantly less activity against SmGGPPS. Several lipophilic bisphosphonates were active against ex vivo adult worms, with worm death occurring over 4 to 6 days. These results indicate that FPPS and GGPPS could be of interest in the context of the emerging resistance to praziquantel in schistosomiasis therapy.

Fig 1

Schematic representation of the mevalonate pathway in Schistosoma mansoni. 3-Hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA), the product of HMG-CoA synthase, is converted to mevalonate by HMG-CoA reductase, the target of the cholesterol-reducing statins. A series of reactions converts mevalonate to dimethylallyl diphosphate (DMAPP), which is converted to its isomer, isopentenyl diphosphate (IPP). Condensations of one DMAPP and one IPP to produce geranyl diphosphate (GPP) and of IPP with GPP to form farnesyl diphosphate (FPP) are carried out by FPP synthase (FPPS) and are the targets of bisphosphonate therapies. Condensation of FPP and IPP leads to the production of geranylgeranyl diphosphate (GGPP) by GGPP synthase (GGPPS). FPP is used to synthesize various quinones, GGPP is used to synthesize dolichols, and both are essential in the posttranslational prenylation of a variety of proteins (e.g., the small GTPases Ras, Rab, and Rho) that are important for cell signaling and survival. Rhodoquinone plays a crucial role in the electron transport chain during cellular respiration in S. mansoni mitochondria. Conversion of FPP to squalene does not occur in S. mansoni, and therefore, there is no cholesterol synthesis.

Fig 2

(A) Structures of substrates and products of SmFPPS and SmGGPPS catalysis. DMAPP, dimethylallyl diphosphate; IPP, isopentenyl diphosphate; GPP, geranyl diphosphate; FPP, farnesyl diphosphate; GGPP, geranylgeranyl diphosphate. (B) Structures of bisphosphonate compounds used in this study.

Fig 3

The most conserved motifs, i.e., the first aspartate-rich motif (FARM) and the second aspartate-rich motif (SARM), in three different types of FPPS and GGPPS enzymes from various species. Residues that form a “lid,” effectively limiting binding to shorter allylic diphosphates, are shown with arrows. Sc, Saccharomyces cerevisiae; Hs, human; Ec, Escherichia coli; Bs, Bacillus stearothermophilus; Tg, Toxoplasma gondii; Pv, Plasmodium vivax; Sm, Schistosoma mansoni; Sa, Sulfolobus acidocaldarius; Mt, Methanobacterium thermoautotrophicum; Pa, Pantoea ananatis; At, Arabidopsis thaliana.

Fig 4

(A) IC₅₀ curves for inhibitor concentration versus percent Schistosoma mansoni FPPS inhibition. (B) IC₅₀ curves for inhibitor concentration versus percent S. mansoni GGPPS inhibition. (C) Survival of ex vivo adult S. mansoni worms cultured in the presence of 50 μM bisphosphonates 2, 91, and 715 as a function of time. (D) Survival of ex vivo adult S. mansoni worms cultured with 50 μM 2, 91, and 715 and with various concentrations of compounds 746, 766, 777, and 820. Worms were cultured in RPMI medium with inhibitors, with the medium and inhibitors replaced daily. Worms were observed for survival (as assessed by motility) over 6 days. The LD₅₀s of the compounds for adult worms are ∼25 μM.