α-Methylation enhances the potency of isoprenoid triazole bisphosphonates as geranylgeranyl diphosphate synthase inhibitors

Disruption of protein geranylgeranylation via inhibition of geranylgeranyl diphosphate synthase (GGDPS) represents a novel therapeutic strategy for a variety of malignancies, especially those characterized by excessive protein secretion such as multiple myeloma. Our work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. Here we present the synthesis and biological evaluation of a new series of isoprenoid triazoles modified by incorporation of a methyl group at the α-carbon. These studies reveal that incorporation of an α-methyl substituent enhances the potency of these compounds as GGDPS inhibitors, and, in the case of the homogeranyl/homoneryl series, abrogates the effects of olefin stereochemistry on inhibitory activity. The incorporation of the methyl group allowed preparation of a POM-prodrug, which displayed a 10-fold increase in cellular activity compared to the corresponding salt. These studies form the basis for future preclinical studies investigating the anti-myeloma activity of these novel α-methyl triazole bisphosphonates.     

Amides as bioisosteres of triazole-based geranylgeranyl diphosphate synthase inhibitors

Geranylgeranyl diphosphate synthase (GGDPS) inhibitors are of potential therapeutic interest as a consequence of their activity against the bone marrow cancer multiple myeloma. A series of bisphosphonates linked to an isoprenoid tail through an amide linkage has been prepared and tested for the ability to inhibit GGDPS in enzyme and cell-based assays. The amides were designed as analogues to triazole-based GGDPS inhibitors. Several of the new compounds show GGDPS inhibitory activity in both enzyme and cell assays, with potency dependent on chain length and olefin stereochemistry.     

Mono- and dialkyl isoprenoid bisphosphonates as geranylgeranyl diphosphate synthase inhibitors

Nitrogenous bisphosphonates are used clinically to reduce bone resorption associated with osteoporosis or metastatic bone disease, and are recognized as inhibitors of farnesyl diphosphate synthase. Inhibition of this enzyme decreases cellular levels of both farnesyl diphosphate and geranylgeranyl diphosphate which results in a variety of downstream biological effects including inhibition of protein geranylgeranylation. Our lab recently has prepared several isoprenoid bisphosphonates that inhibit protein geranylgeranylation and showed that one selectively inhibits geranylgeranyl diphosphate synthase. This results in depletion of intracellular geranylgeranyl diphosphate and impacts protein geranylgeranylation but does not affect protein farnesylation. To clarify the structural features of isoprenoid bisphosphonates that account for their geranylgeranyl diphosphate synthase inhibition, we have prepared a new group of isoprenoid bisphosphonates. The complete set of compounds has been tested for in vitro inhibition of human recombinant geranylgeranyl diphosphate synthase and cellular inhibition of protein geranylgeranylation. These results show some surprising relationships between in vitro and cellular activity, and will guide development of clinical agents directed at geranylgeranyl diphosphate synthase.     

ω-Hydroxy isoprenoid bisphosphonates as linkable GGDPS inhibitors

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple myeloma. Malignant plasma cells produce and secrete large amounts of monoclonal protein, and inhibition of GGDPS results in disruption of protein geranylgeranylation which in turn impairs intracellular protein trafficking. Our previous work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. To explore the possibility of selective delivery of such compounds to plasma cells, new analogues with an ω-hydroxy group have been synthesized and examined for their enzymatic and cellular activity. These studies demonstrate that incorporation of the ω-hydroxy group minimally impairs GGDPS inhibitory activity. Furthermore conjugation of one of the novel ω-hydroxy GGDPS inhibitors to hyaluronic acid resulted in enhanced cellular activity. These results will allow future studies to focus on the in vivo biodistribution of HA-conjugated GGDPS inhibitors.     

Synthesis and biological evaluation of a series of aromatic bisphosphonates

Geminal bisphosphonates display varied biological activity depending on the nature of the substituents on the central carbon atom. For example, the nitrogenous bisphosphonates zoledronate and risedronate inhibit the enzyme farnesyl diphosphate synthase while digeranyl bisphosphonate has been shown to inhibit the enzyme geranylgeranyl diphosphate synthase. We now have synthesized isoprenoid bisphosphonates where an aromatic ring has been used to replace one of the isoprenoid olefins in an isoprenoid bisphosphonate and investigated the ability of these new compounds to impair protein geranylgeranylation within cells. Several of these new compounds are potent inhibitors of the enzyme geranylgeranyl diphosphate synthase.     

Pivaloyloxymethyl-modified isoprenoid bisphosphonates display enhanced inhibition of cellular geranylgeranylation

Nitrogenous bisphosphonate inhibitors of farnesyl disphosphate synthase have been used clinically for treatment of bone disease. Because many of their effects may be mediated by depletion of geranylgeranyl diphosphate, our group has sought compounds that do this more directly through inhibition of geranylgeranyl diphosphate synthase and we have discovered a number of isoprenoid-containing bisphosphonates that selectively inhibit this enzyme. These compounds have a high negative charge at physiological pH which is necessary for inhibition of the enzyme but may limit their ability to enter cells. Therefore, chemical modifications that mask this charge may enhance their cellular potency. We now have synthesized novel pivaloyloxymethyl-modified isoprenoid bisphosphonates and investigated their ability to inhibit protein geranylgeranylation within cells. We have found that addition of pivaloyloxymethyl moieties to isoprenoid bisphosphonates increases their potency towards cellular geranylgeranylation even though this modification decreases their in vitro inhibition of geranylgeranyl diphosphate synthase. Pivaloyloxymethyl modifications more effectively increase the cellular activity of the more polar isoprenoid bisphosphonates. These results reveal structural relationships between in vitro and cellular activity which may serve as the basis for future development of more potent and/or drug-like inhibitors of geranylgeranyl diphosphate synthase.     

Molecular mechanisms linking geranylgeranyl diphosphate synthase to cell survival and proliferation

Geranylgeranyl diphosphate is a 20-carbon isoprenoid phospholipid whose lipid moiety can be post-translationally incorporated into proteins to promote membrane association. The process of geranylgeranylation has been implicated in anti-proliferative effects of clinical agents that inhibit enzymes of the mevalonate pathway (i.e. statins and nitrogenous bisphosphonates) as well as experimental agents that deplete geranylgeranyl diphosphate. Inhibitors of geranylgeranyl diphosphate synthase are an attractive way to block geranylgeranylation because they possess a calcium-chelating substructure to allow localization to bone and take advantage of a unique position of the enzyme within the biosynthetic pathway. Here, we describe recent advances in geranylgeranyl diphosphate synthase expression and inhibitor development with a particular focus on the molecular mechanisms that link geranylgeranyl diphosphate to cell proliferation via geranylgeranylated small GTPases.     

Geranyl and neryl triazole bisphosphonates as inhibitors of geranylgeranyl diphosphate synthase

When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of geranylgeranyl diphosphate synthase.     

Novel benzimidazole phosphonates as potential inhibitors of protein prenylation

Benzimidazole carboxyphosphonates and bisphosphonates have been prepared and evaluated for their activity as inhibitors of protein prenylation or isoprenoid biosynthesis. The nature of the phosphonate head group was found to dictate enzyme specificity. The lead carboxyphosphonate inhibits geranylgeranyl transferase II while its corresponding bisphosphonate analogue potently inhibits farnesyl diphosphate synthase. The most active inhibitors effectively disrupted protein prenylation in human multiple myeloma cells.     

Farnesyl diphosphate synthase; regulation of product specificity

Farnesyl diphosphate synthase (FPPS) is a key enzyme in isoprenoid biosynthesis which supplies sesquiterpene precursors for several classes of essential metabolites including sterols, dolichols, ubiquinones and carotenoids as well as substrates for farnesylation and geranylgeranylation of proteins. It catalyzes the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate. The enzyme is a homodimer of subunits, typically having two aspartate-rich motifs with two sets of substrate binding sites for an allylic diphosphate and isopentenyl diphosphate per homodimer. The synthase amino-acid residues at the 4th and 5th positions before the first aspartate rich motif mainly determine product specificity. Hypothetically, type I (eukaryotic) and type II (eubacterial) FPPSs evolved from archeal geranylgeranyl diphosphate synthase by substitutions in the chain length determination region. FPPS belongs to enzymes encoded by gene families. In plants this offers the possibility of differential regulation in response to environmental changes or to herbivore or pathogen attack.     

Toxoplasma gondii Relies on Both Host and Parasite Isoprenoids and Can Be Rendered Sensitive to Atorvastatin

Intracellular pathogens have complex metabolic interactions with their host cells to ensure a steady supply of energy and anabolic building blocks for rapid growth. Here we use the obligate intracellular parasite Toxoplasma gondii to probe this interaction for isoprenoids, abundant lipidic compounds essential to many cellular processes including signaling, trafficking, energy metabolism, and protein translation. Synthesis of precursors for isoprenoids in Apicomplexa occurs in the apicoplast and is essential. To synthesize longer isoprenoids from these precursors, T. gondii expresses a bifunctional farnesyl diphosphate/geranylgeranyl diphosphate synthase (TgFPPS). In this work we construct and characterize T. gondii null mutants for this enzyme. Surprisingly, these mutants have only a mild growth phenotype and an isoprenoid composition similar to wild type parasites. However, when extracellular, the loss of the enzyme becomes phenotypically apparent. This strongly suggests that intracellular parasite salvage FPP and/or geranylgeranyl diphosphate (GGPP) from the host. We test this hypothesis using inhibitors of host cell isoprenoid synthesis. Mammals use the mevalonate pathway, which is susceptible to statins. We document strong synergy between statin treatment and pharmacological or genetic interference with the parasite isoprenoid pathway. Mice can be cured with atorvastatin (Lipitor) from a lethal infection with the TgFPPs mutant. We propose a double-hit strategy combining inhibitors of host and parasite pathways as a novel therapeutic approach against Apicomplexan parasites.     

An alternative mechanism of product chain-length determination in type III geranylgeranyl diphosphate synthase.

(All-E) prenyl diphosphate synthases catalyze the consecutive condensation of isopentenyl diphosphates with allylic prenyl diphosphates, producing products with various chain-lengths that are unique for each enzyme. Some short-chain (all-E) prenyl diphosphate synthases, i.e. farnesyl diphosphate synthases and geranylgeranyl diphosphate synthases contain characteristic amino acid sequences around the allylic substrate binding sites, which have been shown to play a role in determining the chain-length of the product. However, among these enzymes, which are classified into several types based on the possessive patterns of such characteristics, type III geranylgeranyl diphosphate synthases, which consist of enzymes from eukaryotes (excepting plants), lack these features. In this study, we report that mutagenesis at the second position before the conserved G(Q/E) motif, which is distant from the well-studied region, affects the chain-length of the product for a type III geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae. This clearly suggests that a novel mechanism is operative in the product determination for this type of enzyme. We also show herein that mutagenesis at the corresponding position of an archaeal medium-chain enzyme also alters its product specificity. These results provide valuable information on the molecular evolution of (all-E) prenyl diphosphate synthases.     

rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.

p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic.     

Triazole-based inhibitors of geranylgeranyltransferase II

A small set of triazole bisphosphonates has been prepared and tested for the ability to inhibit geranylgeranyltransferase II (GGTase II). The compounds were prepared through use of click chemistry to assemble a central triazole that links a polar head group to a hydrophobic tail. The resulting compounds were tested for their ability to inhibit GGTase II in an in vitro enzyme assay and also were tested for cytotoxic activity in an MTT assay with the human myeloma RPMI-8226 cell line. The most potent enzyme inhibitor was the triazole with a geranylgeranyl tail, which suggests that inhibitors that can access the enzyme region that holds the isoprenoid tail will display greater activity.     

Cloning of a gene cluster encoding enzymes responsible for the mevalonate pathway from a terpenoid-antibiotic-producing Streptomyces strain

A gene cluster encoding enzymes responsible for the mevalonate pathway was isolated from Streptomyces griseolosporeus strain MF730-N6, a terpenoid-antibiotic terpentecin producer, by searching a flanking region of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene, which had been previously isolated by complementation. By DNA sequencing of an 8.9-kb BamHI fragment, 7 genes encoding geranylgeranyl diphosphate synthase (GGDPS), mevalonate kinase (MK), mevalonate diphosphate decarboxylase (MDPD), phosphomevalonate kinase (PMK), isopentenyl diphosphate (IPP) isomerase, HMG-CoA reductase, and HMG-CoA synthase were suggested to exist in that order. Heterologous expression of these genes in E. coli and Streptomyces lividans, both of which have only the nonmevalonate pathways, suggested that the genes for the mevalonate pathway were included in the cloned DNA fragment. The GGDPS, MK, MDPD, PMK, IPP isomerase, and HMG-CoA synthase were expressed in E. coli. Among them, the recombinant GGDPS, MK, and IPP isomerase were confirmed to have the expected activities. This is the first report, to the best of our knowledge, about eubacterial MK with direct evidence.     

Lysosomal prenylcysteine lyase is a FAD-dependent thioether oxidase

Prenylated proteins contain either a 15-carbon farnesyl or a 20-carbon geranylgeranyl isoprenoid covalently attached via a thioether bond to a cysteine residue at or near their C terminus. As prenylated proteins comprise up to 2% of the total protein in eukaryotic cells, and the thioether bond is a stable modification, their degradation raises a metabolic challenge to cells. A lysosomal enzyme termed prenylcysteine lyase has been identified that cleaves prenylcysteines to cysteine and an unidentified isoprenoid product. Here we show that the isoprenoid product of prenylcysteine lyase is the C-1 aldehyde of the isoprenoid moiety (farnesal in the case of C-15). The enzyme requires molecular oxygen as a cosubstrate and utilizes a noncovalently bound flavin cofactor in an NAD(P)H-independent manner. Additionally, a stoichiometric amount of hydrogen peroxide is produced during the reaction. These surprising findings indicate that prenylcysteine lyase utilizes a novel oxidative mechanism to cleave thioether bonds and provide insight into the unique role this enzyme plays in the cellular metabolism of prenylcysteines.     

Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains

In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography–mass spectrometry. The integration of a geranylgeranyl diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression vector. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. T. Vallon and S. Ghanegaonkar contributed equally to this work.     

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase requires subunit interaction

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase from the bacterium, Pantoea ananatis, was studied. In most types of short-chain (all-E) prenyl diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate-rich motif play a primary role in the product determination mechanism. However, type II geranylgeranyl diphosphate synthase lacks such bulky amino acids at these positions. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechanism of chain length determination in type III geranylgeranyl diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the alpha-helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl diphosphate synthase.     

Mammalian protein geranylgeranyltransferase. Subunit composition and metal requirements.

An enzyme capable of specifically modifying, with a geranylgeranyl isoprenoid, candidate proteins containing a consensus prenylation sequence ending in leucine has been purified from bovine brain. This protein geranylgeranyltransferase (PGGT), isolated using affinity chromatography on an immobilized peptide column, contains two subunits with molecular masses of 48 and 43 kDa, designated alpha and beta, respectively. An antiserum raised to the alpha subunit of the related enzyme, protein farnesyltransferase (PFT), also recognizes this chromatographically identical alpha-subunit of the PGGT by immunoblot analysis. The PGGT and PFT enzymes from bovine brain are shown to be dependent on both Mg2+ and Zn2+ for optimal activity. Demonstration of the Zn2+ dependence of the enzymes requires prolonged incubation or purification in the presence of a chelating agent; we therefore propose that these enzymes be placed into the category of metalloenzymes. Under optimal assay conditions, these enzymes show high specificity toward their prenyl diphosphate substrates, with only a weak competition observed with farnesyl diphosphate in the PGGT reaction or geranylgeranyl diphosphate in the PFT reaction. The two enzymes are differentially sensitive to several detergents tested to determine suitable ones for product stabilization in the reactions. These results confirm previous predictions on the subunit structure of the PGGT and provide an avenue to initiating a molecular analysis of the geranylgeranyl modification of many mammalian proteins.     

Directed evolution of metabolically engineered Escherichia coli for carotenoid production

We have previously introduced a reconstructed isoprenoid pathway into Escherichia coli that exhibits amplified biosynthetic flux to geranylgeranyl diphosphate (GGPP), a common isoprenoid precursor. It was shown that GGPP synthase is an important rate-controlling enzyme in this reconstructed isoprenoid pathway. In this investigation, we applied directed evolution to GGPP synthase from Archaeoglobus fulgidus to enable the enhanced production of carotenoids in metabolically engineered E. coli. Eight mutants were isolated, and the best one increased lycopene production by 100%. Among the mutants that were isolated, mutation points were clustered in four "hot regions". The "hottest" region is located in the sequence upstream of the coding region, which presumably improves the expression level of the enzyme. The other three are within the coding sequence and are believed to improve the enzyme-specific activity in E coli. These results demonstrate that modulating both enzymatic expression and specific activity are important for optimizing the metabolic flux distribution.