Lipophilic analogs of zoledronate and risedronate inhibit Plasmodium geranylgeranyl diphosphate synthase (GGPPS) and exhibit potent antimalarial activity

Abstract

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 ½Mg 2þ 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.M alaria, caused by Plasmodium spp., causes approximately 1 million deaths each year (1), and there are ever-present problems due to drug resistance (2). There is, therefore, a need for new drugs and drug leads. In earlier work, we and others found that the bisphosphonate class of drugs (3) used to treat bonerelated diseases-osteoporosis, Paget disease, and hypercalcemia due to malignancy-also inhibited the growth of a range of parasitic protozoa, including Trypanosoma cruzi (4, 5), Trypanosoma brucei (4, 6), Leishmania spp. (4,7,8), Toxoplasma gondii (4, 9), Cryptosporidium parvum (10, 11), Entamoeba histolytica (4, 12, 13), and Plasmodium spp. (4, 13-15). In the case of Plasmodium spp., the most potent inhibitors were not, however, the nitrogencontaining bisphosphonates such as zoledronate or risedronate (Scheme 1) used to treat bone diseases, but more lipophilic n-alkyl bisphosphonates (13). Their target in Plasmodium falciparum was not determined. However, more recently, a Plasmodium vivax geranylgeranyl diphosphate synthase (PvGGPPS) has been cloned, expressed, purified, and crystallized, and its three-dimensional structure determined (16). The enzyme is inhibited by bisphosphonates (16), so it seemed possible that it might be a target for the inhibitors discovered earlier. To investigate this possibility, we recently determined the IC 50 values for 25 bisphosphonates against PvGGPPS and compared the results for enzyme inhi...

Isoprenoid Biosynthesis in Plasmodium falciparum

“…In P. falciparum, the condensation of GPP and IPP (to produce FPP) and of FPP and IPP (to produce geranylgeranyl diphosphate [GGPP]) appears to be catalyzed by a bifunctional FPP-GGPP synthase (PlasmoDB ID PF3D7_1128400) (49). The crystal structure of the P. vivax enzyme has been solved (50,51). This bifunctional enzyme is sensitive to bisphosphonates, such as zoledronate and risedronate (49).…”

Bisphosphonate Inhibitors Reveal a Large Elasticity of Plastidic Isoprenoid Synthesis Pathway in Isoprene-Emitting Hybrid Aspen

“…3; Tables I and II), indicating a noncompetitive or irreversible inhibition. Existence of such a direct inhibition could be expected given that, in the case of inhibition of GDP, FDP, and GGDP synthases, alendronate and zoledronate bind to the three-Mg 2+ cluster in the allylbinding (DMADP-binding) pocket of the enzyme active site (Jahnke et al, 2010;No et al, 2012;Lindert et al, 2013;Liu et al, 2014). The active site of isoprene synthase also coordinates the diphosphate tail of DMADP by three Mg 2+ atoms (Köksal et al, 2010), and is thus analogous to the allyl-binding active site in prenyltransferases.…”

“…They have been demonstrated to inhibit cytosolic farnesyl diphosphate (FDP) synthase activity (Oberhauser et al, 1998;Cromartie et al, 1999;van Beek et al, 1999;Bergstrom et al, 2000;Burke et al, 2004), аs well as geranyl diphosphate (GDP) and geranylgeranyl diphosphate (GGDP) synthase activities (Oberhauser et al, 1998;Cromartie et al, 1999;Kloer et al, 2006;No et al, 2012;Lindert et al, 2013). To our knowledge, bisphosphonates have not been used to study the effects of end product accumulation on the pathway flux in isopreneemitting species, with the exception of one study that investigated the development of isoprene emission capacity through leaf ontogeny (Rasulov et al, 2014).…”

Restricted Substrate Specificity for the Geranylgeranyltransferase-I Enzyme in Cryptococcus neoformans: Implications for Virulence

“…More recently, a number of these prenyltransferase inhibitors have been repurposed to inhibit eukaryotic pathogens. Prenyltransferase inhibitors have been shown to be effective against two pathogenic parasites, Trypanosoma brucei and Plasmodium falciparum, in mouse models of infection (12)(13)(14). Additionally, prenyltransferases have been shown to be important for fungal pathogen virulence and virulence-associated phenotypes.…”