Human Geranylgeranyl Diphosphate Synthase Research Articles

Moiety-Linkage Map Reveals Selective Nonbisphosphonate Inhibitors of Human Geranylgeranyl Diphosphate Synthase

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS). Current bisphosphonate drugs (e.g., Fosamax and Zometa) are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease, and tumor-induced osteolysis, but they are often less potent in blood and soft-tissue due to their phosphate moieties. The discovery of nonbisphosphonate inhibitors of FPPS and/or GGPPS for the treatment of bone diseases and cancers is, therefore, a current goal. Here, we propose a moiety-linkage-based method, combining a site-moiety map with chemical structure rules (CSRs), to discover nonbisphosphonate inhibitors from thousands of commercially available compounds and known crystal structures. Our moiety-linkage map reveals the binding mechanisms and inhibitory efficacies of 51 human GGPPS (hGGPPS) inhibitors. To the best of our knowledge, we are the first team to discover two novel selective nonbisphosphonate inhibitors, which bind to the inhibitory site of hGGPPS, using CSRs and site-moiety maps. These two compounds can be considered as a novel lead for the potent inhibitors of hGGPPS for the treatment of cancers and mevalonate-pathway diseases. Moreover, based on our moiety-linkage map, we identified two key residues of hGGPPS, K202, and K212, which play an important role for the inhibitory effect of zoledronate (IC50 = 3.4 μM and 2.4 μM, respectively). This result suggests that our method can discover specific hGGPPS inhibitors across multiple prenyltransferases. These results show that the compounds that highly fit our moiety-linkage map often inhibit hGGPPS activity and induce tumor cell apoptosis. We believe that our method is useful for discovering potential inhibitors and binding mechanisms for pharmaceutical targets.

Structures of a potent phenylalkyl bisphosphonate inhibitor bound to farnesyl and geranylgeranyl diphosphate synthases

We report the X-ray crystallographic structures of the bisphosphonate N-[methyl(4-phenylbutyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonate (BPH-210), a potent analog of pamidronate (Aredia), bound to farnesyl diphosphate synthase (FPPS) from Trypanosoma brucei as well as to geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae. BPH-210 binds to FPPS, together with 3 Mg(2+), with its long, hydrophobic phenylbutyl side-chain being located in the same binding pocket that is occupied by allylic diphosphates and other bisphosphonates. Binding is overwhelmingly entropy driven, as determined by isothermal titration calorimetry. The structure is of interest since it explains the lack of potency of longer chain analogs against FPPS, since these would be expected to have a steric clash with an aromatic ring at the distal end of the binding site. Unlike shorter chain FPPS inhibitors, such as pamidronate, BPH-210 is also found to be a potent inhibitor of human geranylgeranyl diphosphate synthase. In this case, the bisphosphonate binds only to the GGPP product inhibitory site, with only 1 (chain A) or 0 (chain B) Mg(2+), and DeltaS is much smaller and DeltaH is approximately 6 k cal more negative than in the case of FPPS binding. Overall, these results are of general interest since they show that some bisphosphonates can bind to more than one trans-prenyl synthase enzyme which, in some cases, can be expected to enhance their overall activity in vitro and in vivo.

Human Geranylgeranyl Diphosphate Synthase is an Octamer in Solution

A recombinant geranylgeranyl diphosphate synthase (GGPS) was analysed to be a mixture of octamer, hexamer and dimer by gel filtration using a Superdex 200 column followed by the blue native polyacrylamide gel electrophoresis. The hexamer and dimer were each converted to an octamer by treating with dithiothreitol (DTT). When the recombinant GGPS was preliminarily treated with DTT and similarly analysed, octamer was predominantly detected with a trace amount of hexamer. The octameric form of GGPS was also supported by the cross-linking experiments with bis(sulfosuccinimidyl) suberate. The GGPS in an octameric form was active with a combination of farnesyl diphosphate and [1-(14)C]isopentenyl diphosphate. These results indicate that the active form of GGPS in the solution is an octamer rather than hexamer or dimer.

Gerfelin, a novel inhibitor of geranylgeranyl diphosphate synthase from Beauveria felina QN22047. I. Taxonomy, fermentation, isolation, and biological activities.

A new compound, gerfelin, was isolated from a culture broth of Beauveria felina QN22047. It was purified by column chromatography on silica gel and by HPLC. Gerfelin has the molecular formula C15H14O6. It inhibited synthesis of geranylgeranyl diphosphate, which was mediated by recombinant human geranylgeranyl diphosphate synthase (hGGPP synthase) in vitro. The inhibitory pattern of gerfelin was noncompetitive against isopentenyl diphosphate, and uncompetitive against farnesyl diphosphate.

Human Geranylgeranyl Diphosphate Synthase: cDNA CLONING AND EXPRESSION

Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C20-prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR.RXR). The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed in Escherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase. Northern blot analysis indicates the presence of two forms of the mRNA.

Human geranylgeranyl diphosphate synthase: isolation of the cDNA, chromosomal mapping and tissue expression

We report the nucleotide sequence of human geranylgeranyl diphosphate (GGPP) synthase cDNA isolated from a fetal heart library. The 2.5 kb cDNA encodes a protein of 34 kDa. The protein contains six domains that have been identified previously in many other prenyltransferases. Recombinant, purified histidine-tagged protein exhibited the enzymatic properties associated with GGPP synthase, namely the synthesis of GGPP from farnesyl diphosphate and isopentenyl diphosphate. Transient transfection of mammalian cells with a plasmid encoding the putative GGPP synthase resulted in a 55-fold increase in GGPP synthase activity. Taken together, these results establish that the cDNA encodes the mammalian GGPP synthase protein. The mRNA for GGPP synthase was expressed ubiquitiously. Of the 16 human tissues examined, the highest expression of the mRNA was in testis. The mRNA levels in cultured HeLa cells were unaffected by alterations in cellular sterol levels and contrasted with the significant regulation of isopentenyl diphosphate synthase mRNA under these same conditions. Fluorescent in situ hybridization was used to map the single gene encoding human GGPP synthase to chromosome 1q43.—Ericsson, J., J. M. Greene, K. C. Carter, B. K. Shell, D. R. Duan, C. Florence, and P. A. Edwards. Human geranylgeranyl diphosphate synthase: isolation of the cDNA, chromosomal mapping, and tissue expression.