Enzyme Geranylgeranyl Diphosphate Synthase Research Articles

Deficiency of geranylgeranyl biphosphate synthase in kidney tubules causes cystic kidney disease.

Polycystic kidney disease (PKD) is a common hereditary kidney disease. Although PKD occurrence is associated with certain gene mutations, its onset regulatory mechanisms are still not well understood. Here, we first report that the key enzyme geranylgeranyl diphosphate synthase (GGPPS) is specifically expressed in renal tubular epithelial cells of mouse kidneys. We aimed to explore the role of GGPPS in PKD. In this study, we established a Ggppsfl/fl:Cdh16cre mouse model and compared its phenotype with that of wild-type mice. A Ggpps-downregulation HK2 cell model was also used to further determine the role of GGPPS. We found that GGPPS was specifically expressed in renal tubular epithelial cells of mouse kidneys. Its expression also increased with age. Low GGPPS expression was observed in human ADPKD tissues. In the Ggppsfl/fl:Cdh16cre mouse model, Ggpps deletion in renal tubular epithelial cells induced the occurrence and development of renal tubule cystic dilation and caused the death of mice after birth due to abnormal renal function. Enhanced proliferation of cyst-lining epithelial cells was also observed after the knockout of Ggpps. These processes were related to the increased rate of Rheb on membrane/cytoplasm and hyperactivation of mTORC1 signaling. In conclusion, the deficiency of GGPPS in kidney tubules induced the formation of renal cysts. It may play a critical role in PKD pathophysiology. A novel therapeutic strategy could be designed according to this work.

α-Amino bisphosphonate triazoles serve as GGDPS inhibitors

Depletion of cellular levels of geranylgeranyl diphosphate by inhibition of the enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential strategy for disruption of protein transport by limiting the geranylgeranylation of the Rab proteins that regulate intracellular trafficking. As such, there is interest in the development of GGDPS inhibitors for the treatment of malignancies characterized by abnormal protein production, including multiple myeloma. Our previous work has explored the structure-function relationship of a series of isoprenoid triazole bisphosphonate-based GGDPS inhibitors, with modifications having impact on enzymatic, cellular and in vivo activities. We have synthesized a new series of α-amino bisphosphonates to understand the impact of modifying the alpha position with a moiety that is potentially linkable to other agents. Bioassays evaluating the enzymatic and cellular activities of these compounds demonstrate that incorporation of the α-amino group affords compounds with GGDPS inhibitory activity which is modulated by isoprenoid tail chain length and olefin stereochemistry. These studies provide further insight into the complexity of the structure-function relationship and will enable future efforts focused on tumor-specific drug delivery.

Evaluation of geranylgeranyl diphosphate synthase inhibition as a novel strategy for the treatment of osteosarcoma and Ewing sarcoma.

Rab GTPases are critical regulators of protein trafficking in the cell. To ensure proper cellular localization and function, Rab proteins must undergo a posttranslational modification, termed geranylgeranylation. In the isoprenoid biosynthesis pathway, the enzyme geranylgeranyl diphosphate synthase (GGDPS) generates the 20-carbon isoprenoid donor (geranylgeranyl pyrophosphate [GGPP]), which is utilized in the prenylation of Rab proteins. We have pursued the development of GGDPS inhibitors (GGSI) as a novel means to target Rab activity in cancer cells. Osteosarcoma (OS) and Ewing sarcoma (ES) are aggressive childhood bone cancers with stagnant survival statistics and limited treatment options. Here we show that GGSI treatment induces markers of the unfolded protein response (UPR) and triggers apoptotic cell death in a variety of OS and ES cell lines. Confirmation that these effects were secondary to cellular depletion of GGPP and disruption of Rab geranylgeranylation was confirmed via experiments using exogenous GGPP or specific geranylgeranyl transferase inhibitors. Furthermore, GGSI treatment disrupts cellular migration and invasion in vitro. Metabolomic profiles of OS and ES cell lines identify distinct changes in purine metabolism in GGSI-treated cells. Lastly, we demonstrate that GGSI treatment slows tumor growth in a mouse model of ES. Collectively, these studies support further development of GGSIs as a novel treatment for OS and ES.

Impact of α-modifications on the activity of triazole bisphosphonates as geranylgeranyl diphosphate synthase inhibitors

Agents that inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS) have anti-cancer activity and our prior studies have investigated the structure-function relationship for a family of isoprenoid triazole bisphosphonates as GGDPS inhibitors. To further explore this structure-function relationship, a series of novel α-modified triazole phosphonates was prepared and evaluated for activity as GGDPS inhibitors in enzyme and cell-based assays. These studies revealed flexibility at the α position of the bisphosphonate derivatives with respect to being able to accommodate a variety of substituents without significantly affecting potency compared to the parent unsubstituted inhibitor. However, the monophosphonate derivatives lacked activity. These studies further our understanding of the structure-function relationship of the triazole-based GGDPS inhibitors and lay the foundation for future studies evaluating the impact of α-modifications on in vivo activity.

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.

In Vivo Evaluation of Isoprenoid Triazole Bisphosphonate Inhibitors of Geranylgeranyl Diphosphate Synthase: Impact of Olefin Stereochemistry on Toxicity and Biodistribution.

The enzyme geranylgeranyl diphosphate synthase (GGDPS) synthesizes the 20-carbon isoprenoid geranylgeranyl pyrophosphate, which is used in geranylgeranylation reactions. We have demonstrated that GGDPS inhibitors in multiple myeloma (MM) cells disrupt Rab geranylgeranylation, leading to inhibition of monoclonal protein trafficking, induction of the unfolded protein response pathway (UPR), and apoptosis. We have previously reported preclinical studies with the GGDPS inhibitor VSW1198, which is a mixture of homogeranyl/homoneryl triazole bisphosphonates. Additional structure-function efforts have led to development of the α-methylated derivatives RAM2093 (homogeranyl) and RAM2061 (homoneryl). As little is known regarding the impact of olefin stereochemistry on drug properties in vivo, we pursued additional preclinical evaluation of RAM2093 and RAM2061. In MM cell lines, both isomers induce activation of UPR/apoptotic markers in a concentration-dependent manner and with similar potency. Single-dose testing in CD-1 mice identified a maximum tolerated i.v. dose of 0.5 mg/kg for RAM2061 and 0.3 mg/kg for RAM2093. Liver toxicity was the primary barrier to dose escalation for both compounds. Disruption of geranylgeranylation in vivo was confirmed after multidose administration of either compound. Pharmacokinetic studies revealed plasma terminal half-lives of 29.2 ± 6 (RAM2061) and 22.1 ± 4 hours (RAM2093). Relative to RAM2061, RAM2093 levels were significantly higher in liver tissue but not in other tissues. Using MM.1S flank xenografts, we observed a significant reduction in tumor growth in mice treated with RAM2061 relative to controls. Collectively, these studies reveal olefin stereochemistry-dependent effects on GGDPS inhibitor biodistribution and confirm the in vivo efficacy of this novel therapeutic approach. SIGNIFICANCE STATEMENT: These studies reveal olefin stereochemistry-dependent effects on the in vivo properties of two novel triazole bisphosphonate inhibitors of geranylgeranyl diphosphate synthase and demonstrate the therapeutic potential of this class of inhibitors for the treatment of multiple myeloma.

In Vivo Evaluation of Novel Geranylgeranyl Diphosphate Synthase Inhibitors

▪The enzyme geranylgeranyl diphosphate synthase (GGDPS) synthesizes the 20-carbon isoprenoid geranylgeranyl diphosphate which is used in protein geranylgeranylation reactions. Our work has demonstrated that GGDPS inhibitors (GGDPSIs) represent a novel therapeutic strategy for multiple myeloma (MM) by disrupting Rab protein geranylgeranylation. Treatment of MM cells with GGDPSI results in disruption of monoclonal protein trafficking, leading to induction of the unfolded protein response pathway (UPR) and apoptosis. We have previously reported preclinical studies with a lead GGDPSI, VSW1198 (a mixture of homogeranyl/homoneryl triazole bisphosphonates), demonstrating the agent's metabolic stability, prolonged half-life (plasma elimination half-life of 47.7 (±7.4) hrs), systemic distribution and confirmed in vivo disruption of geranylgeranylation (Haney et al., Invest New Drugs, 2018). Additional structure-function efforts have led to the development of the α-methylated derivatives RAM2093 (homogeranyl) and RAM2061 (homoneryl). Intriguingly the addition of the α-methyl group abrogates the effects of the olefin stereochemistry on inhibitor potency such that the individual isomers display near identical ability to disrupt protein geranylgeranylation in enzyme and cell assays (Matthiesen et al., Bioorg Med Chem, 2018). As little is known regarding the impact of olefin stereochemistry on the pharmacokinetic (PK)/pharmacodynamic (PD) properties of drugs, we pursued additional in vitro and in vivo studies of RAM2093 and RAM2061 and investigated the efficacy of the GGDPSIs in a mouse MM xenograft model. In MM cell lines, qRT-PCR and western blot analysis showed that both isomers induce activation of UPR/apoptotic markers in a dose-dependent manner and with similar potency. Single dose testing in CD-1 mice identified a maximum tolerated dose of 0.5 mg/kg IV for RAM2061 and 0.3 mg/kg for RAM2093. Liver toxicity was the primary barrier to dose escalation with both compounds. Analysis of blood samples showed elevated liver transaminase levels with normal bilirubin/alkaline phosphatase levels and histopathological examination confirmed evidence of hepatocyte injury at higher doses. Multi-dose schedules of 0.1 mg/kg twice a week (two weeks on, one week off, two weeks on) for RAM2061 and RAM2093, as well as 0.2 mg/kg (RAM2061) and 0.15 mg/kg (RAM2093) weekly x four weeks were tested. All dosing schedules were tolerated with the exception of the twice-weekly schedule for RAM2093. Consistent with the findings from our single dose testing, both multi-dose schedules induced transient elevation of hepatic transaminases. No loss of weight was observed and creatinine and CBC results were within normal limits for both single and multi-dose injected animals. Importantly, western blot analysis of mouse tissues collected from both RAM2061 and RAM2093 multi-dose-treated mice showed accumulation of unmodified Rap1a in the liver, kidney and spleen, indicating in vivo disruption of protein geranylgeranylation was achieved. Furthermore, using MM.1S MM cell flank xenographs in NOD-SCID mice, we observed a significant reduction in tumor growth in mice treated with either VSW1198 or RAM2061 relative to vehicle control. Lastly, PK/biodistribution studies with RAM2093 and RAM2061 were performed following a single dose of 0.3 mg/kg IV. Both compounds were detectable in plasma and liver samples for up to seven days post-injection demonstrating prolonged half-life and tissue distribution. The full PK parameters for both compounds will be presented and studies measuring drug levels in other tissues are ongoing. Taken together, these data suggest that RAM2061 and RAM2093 have equivalent anti-MM activity in vitro, but that RAM2061 is better tolerated in vivo. Whether this is a consequence of different PK properties vs. differences in tissue uptake or metabolism will be determined. These studies also confirm the in vivo efficacy of our novel GGDPSIs and support further development of these agents for the treatment of MM. [Display omitted] DisclosuresHolstein:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Isoprenoid Amide Bisphosphonates As a Novel Class of Geranylgeranyl Diphosphate Synthase Inhibitors

Abstract Multiple myeloma is characterized by the production of monoclonal protein by clonal plasma cells and is a malignancy inherently susceptible to agents that disrupt protein homeostasis. We have previously demonstrated that targeting the geranylgeranylation of Rab GTPases is a novel mechanism by which to induce myeloma cell apoptosis via disruption of intracellular monoclonal protein trafficking and induction of the unfolded protein response pathway. Our previous efforts have focused on the development of novel triazole isoprenoid bisphosphonates as inhibitors of the enzyme geranylgeranyl diphosphate synthase (GGDPS). GGDPS is responsible for the synthesis of the 20-carbon isoprenoid (GGDP) that is utilized in protein geranylgeranylation reactions. Extensive structure-function analysis of the triazole-based inhibitors has revealed that both chain length and olefin stereochemistry have significant impact on inhibitor potency. Previous reports have utilized triazoles as bioisoteres of amides and we hypothesized that the converse might be true as well; namely that amides analogous to the triazoles would have efficacy as GGDPS inhibitors. To this end, we have prepared a number of novel isoprenoid amide bisphosphonates as potential GGDPS inhibitors. These new compounds were prepared from bisphosphonate 1 through a short reaction sequence including chain extension by conjugate addition of nitromethane, reduction to the amine 2, amide formation through reaction with a variety of carboxylic acids, and final hydrolysis of the phosphonate esters to obtain the salts 3. These analogues differed with respect to chain length (ranging from bishomoprenyl- to bishomogeranyl-length), olefin stereochemistry (E vs Z), absence of the proximal olefin (i.e., citronellyl) as well as the stereochemistry of the citronellyl derivatives (R vs S). All of the novel compounds were subjected to in vitro enzyme assays with GGDPS as well as for the related enzymes farnesyl diphosphate synthase, farnesyl transferase and geranylgeranyl transferase I, and IC50's were determined. Cellular activity in human myeloma cell lines (RPMI-8226, MM.1S) was assessed via immunoblot analysis of representative prenylated proteins. In addition, ELISA of intracellular levels of lambda light chain (a marker of disruption of Rab geranylgeranylation) was performed. These studies reveal that similar to the previously reported triazoles, both chain length and olefin stereochemistry have significant effects on GGDPS inhibitory activity. Extending the chain length from the bishomoprenyl length to the bishomogeranyl length increased cellular activity 50-fold: the lowest effective concentration (LEC) is 50 μM for the bishomoprenyl compound and 1 μM for the bishomogeranyl compound. Furthermore, changing the olefin stereochemistry to the bishomoneryl form (i.e., the Z-configuration), further enhanced the potency to an LEC of 0.5 μM. Interestingly, the stereochemistry of the citronellyl derivatives were found to have a significant impact on activity with the R-enantiomer being 10-fold more potent than the S-enantiomer (LEC of 5 μM vs 50 μM respectively). Enzyme assay studies confirmed the specificity of these agents as GGDPS inhibitors. In conclusion, these studies establish a novel family of amide isoprenoid bisphosphonates as GGDPS inhibitors with activity against myeloma cells. Further structure-function analysis will better define the optimal chain length and stereochemistry to maximize potency and enable future in vivo studies assessing efficacy as anti-myeloma therapy. Figure. Figure. Disclosures Wiemer: Terpenoid Therapeutics, Inc: Other: Founder. Holstein:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Recent Advances in the Development of Mammalian Geranylgeranyl Diphosphate Synthase Inhibitors.

The enzyme geranylgeranyl diphosphate synthase (GGDPS) catalyzes the synthesis of the 20-carbon isoprenoid geranylgeranyl diphosphate (GGPP). GGPP is the isoprenoid donor for protein geranylgeranylation reactions catalyzed by the enzymes geranylgeranyl transferase (GGTase) I and II. Inhibitors of GGDPS result in diminution of protein geranylgeranylation through depletion of cellular GGPP levels, and there has been interest in GGDPS inhibitors as potential anti-cancer agents. Here we discuss recent advances in the development of GGDPS inhibitors, including insights gained by structure-function relationships, and review the preclinical data that support the continued development of this novel class of drugs.

Synergistic Inhibition of Geranylgeranyl Diphosphate Synthase By a Mixture of Olefin Stereoisomers

The production of monoclonal protein (MP) by malignant plasma cells is a hallmark of multiple myeloma (MM). We have previously demonstrated that targeting the Rab GTPase family via disruption of the post-translational modification geranylgeranylation impairs intracellular monoclonal protein trafficking, leading to endoplasmic reticulum stress and apoptosis. Thus, inhibition of Rab geranylgeranylation represents a novel therapeutic strategy in MM. One approach by which to disrupt protein geranylgeranylation is through inhibition of the enzyme geranylgeranyl diphosphate synthase (GGDPS), which catalyzes the synthesis of the 20-carbon geranylgeranyl diphosphate (GGDP) from the 15-carbon farnesyl diphosphate (FDP) and the 5-carbon isopentenyl diphosphate (IPP). We have previously reported a series of triazole bisphosphonates and their activity as GGDPS inhibitors. The lead compound in this series was found to be the triazole 1 which was initially prepared as a mixture of isomers in a 3:1 ratio of the homogeranyl:homoneryl isomers and was found to be the most potent GGDPS inhibitor published to date with an IC50 of 45 nM. Our prior work involving geranyl and neryl triazoles had revealed that the neryl isomer was significantly more potent than the geranyl and that the mix of those two isomers had intermediate activity. We therefore hypothesized that the homoneryl isomer 2 would prove to be more potent than the homogeranyl isomer 3. To this end, each individual isomer was prepared in pure form and subjected to in vitro enzyme assays for GGDPS and the related enzyme FDPS. Cellular activity in human myeloma cell lines (RPMI-8226, MM.1S) was assessed via immunoblot analysis of unmodified Rap1a (as a representative geranylgeranylated protein) as well as ELISA of intracellular levels of lambda light chain (as a marker for disruption of Rab geranylgeranylation). As shown in Table 1, while 2 was found to be a more potent inhibitor of GGDPS than 3, interestingly, it was less potent than the original 3:1 mixture (1). The isomers only weakly inhibited FDPS, revealing specificity for GGDPS. Immunoblot analysis and ELISA studies also demonstrated that 2 more potently disrupts cellular geranylgeranylation than its olefin isomer 3. In addition, measurement of intracellular GGPP levels confirmed that 2 more potently decreased cellular GGPP levels than 3. To investigate why the individual isomers were less potent than the original mixture, studies were performed in which cells were treated with varying concentrations of 2 and 3 both alone and in different combinations. These studies revealed a synergistic increase in both unmodified Rap1a and intracellular light chains when the cells were treated with a 3:1 mixture of 3 and 2, respectively. However, no such increase was observed with either a 1:3 mixture or a 1:1 mixture. Notably, this synergy between the two isomers was also observed in the GGDPS enzyme assay (combination index for EC50 of 0.25), suggesting that the two isomers might bind simultaneously to the active site but within different domains. Using computer modeling and available crystallography data, it was revealed that 2 is preferred at the FPP site and binds with very high affinity while 3 demonstrates a preference for the GGPP site and that both may bind to the enzyme simultaneously. Crystallographic studies involving the protein and the isomer mixture are now underway to confirm the results of the modeling studies. The activity of 2 and 3, both alone and in combination could be further potentiated by co-incubation with submicromolar concentrations of lovastatin, an HMG-CoA reductase inhibitor. This has clinical significance as submicromolar levels of statins are readily achieved with standard therapeutic dosing and thus could be used to enhance activity of the novel drugs in vivo. In conclusion, these results are highly intriguing as they demonstrate olefin stereoisomers that have synergistic inhibitory activity against the target enzyme GGDPS. These studies establish homogeranyl/homoneryl triazole bisphosphonate 1 as the lead GGDPS inhibitor as a potential therapeutic agent for multiple myeloma and provide new directions for future development of inhibitors which are designed to simultaneously interact with multiple binding sites within the target enzyme. [Display omitted] [Display omitted] DisclosuresWiemer:Terpenoid Therapeutics, Inc.: Other: Founder. Holstein:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees.

Stereoselective Synthesis of Homoneryl and Homogeranyl Triazole Bisphosphonates.

Isoprenoid-substituted bisphosphonates are known to serve as inhibitors of the enzyme geranylgeranyl diphosphate synthase, and their activity can be highly sensitive to olefin stereochemistry. A mixture of homogeranyl and homoneryl triazole bisphosphonates has previously demonstrated potent activity, and thus stereocontrolled syntheses of the individual isomers have been developed.

Targeting protein geranylgeranylation slows tumor development in a murine model of prostate cancer metastasis

ABSTRACTThe isoprenoid biosynthetic pathway (IBP) plays a critical role in providing substrates and enzymes necessary for the post-translational modification and thus activation of a number of proteins involved in prostate cancer metastasis. Previous work by our lab found novel compound disodium [(6Z,11E,15E)-9-[bis(sodiooxy)phosphoryl]−17-hydroxy-2,6,12,16-tetramethyheptadeca-2,6,11,15-tetraen-9-yl]phosphonate (GGOHBP), which inhibits the IBP enzyme geranylgeranyl diphosphate synthase (GGDPS), reduced protein geranylgeranylation without altering protein farnesylation. This activity significantly reduced adrenal gland tumor burden in a murine model of human prostate cancer metastasis which relied on treatment of established disease. The present study determined the ability of GGDPS inhibition to slow the development of prostate cancer metastasis in a preventative murine model. Using tail vein injection of human derived PC-3 prostate cancer cells 4 d after initiating daily GGOHBP or vehicle treatments, we found GGOHBP significantly reduced whole body tumor burden, significantly slowed the development of tumors, and prolonged overall survival as compared to vehicle treated animals. The observed reduction in soft tissue tumor burden corresponded to a biochemical reduction in Rap1A geranylgeranylation, which for prostate cancer is important in its own merit and which serves as a surrogate marker for Rho family, i.e. Rac, protein modification. This effect was present in all treated mice pointing to strong target engagement, which was not observed in non-tumor burdened tissues or control mice. Our findings reiterate a role for protein geranylgeranylation in the development of prostate cancer metastasis in vivo.

A new motif for inhibitors of geranylgeranyl diphosphate synthase

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure–function relationship which is dependent on the nature of the alkyl group at the α-carbon.

A Novel Class of Geranylgeranyl Diphosphate Synthase Inhibitors: Structure-Activity Relationships of Homoisoprenoid Triazoles in Myeloma Cells

The secretion of monoclonal protein and the dependence on the surrounding bone marrow microenvironment are key features of malignant plasma cells. We have previously demonstrated that targeting the Rab family of small GTPases via disruption of the post-translational modification geranylgeranylation not only impairs intracellular monoclonal protein trafficking, thus leading to endoplasmic reticulum stress and apoptosis, but also disrupts the interaction of the malignant plasma cells with key components of the bone marrow microenvironment. While developing agents that directly target the enzyme that geranylgeranylates the Rabs, we discovered several compounds that specifically and potently inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS). This enzyme is responsible for synthesis of the isoprenoid substrate which is used in geranylgeranylation reactions, thus inhibition of this enzyme is an alternative approach by which to target Rabs. Families of compounds were therefore designed which incorporated a hydrophobic chain, a triazole ring, and a bisphosphonic acid head group. The central triazole provides a potential zinc-binding element, and allows for divergent assembly of potential inhibitors through click chemistry. Through this strategy, prenyl, homoprenyl, geranyl, homogeranyl, farnesyl, homofarnesyl, geranylgeranyl, and homogeranylgeranyl triazole bisphosphonates have been prepared. All of these agents were subjected to in vitro enzyme assays for GGDPS as well as for the related enzyme farnesyl diphosphate synthase (FDPS), and IC50’s were determined. Cellular activity in human myeloma cell lines (RPMI-8226, U266, MM.1S) was assessed via immunoblot analysis of representative prenylated proteins. These studies revealed that in all cases, the homologated-version of the allylic triazole is more potent than the corresponding isoprenoid against GGDPS, ranging from 1.1-fold improvement in the homogeranylgeranyl:geranylgeranyl pairing to almost 300-fold in the homogeranyl:geranyl pairing. Chain length was found to be an important factor which determines the inhibitory activity within the homoisoprenoid series: the order of increasing potency against GGDPS was homogeranylgeranyl < homofarnesyl < homoprenyl << homogeranyl. The latter compound was determined to be the lead compound in this series with an IC50 of 58 nM against GGDPS and 480-fold selectivity for GGDPS compared with FDPS. Disruption of protein geranylgeranylation in myeloma cells with accompanying accumulation of intracellular monoclonal protein levels was observed at concentrations as low as 100 nM. This activity could be further potentiated with co-incubation with submicromolar concentrations of lovastatin, an HMG-CoA reductase inhibitor. Co-incubation studies with the isoprenoid intermediates mevalonate, farnesyl diphosphate and geranylgeranyl diphosphate (GGPP) revealed that the cellular activity of the homogeranyl triazole bisphosphonate is due to depletion of GGPP, consistent with the activity seen in the enzyme assays. Further studies demonstrated that this agent, in a concentration-dependent manner, induces elements of the unfolded protein response pathway and disrupts autophagy, as determined by immunoblot analysis of calnexin cleavage, levels of phosphorylated eIF2a, and LC3 cleavage. Treatment of human bone marrow stromal cells (HS-5 cell line) with the lead compound induced a concentration-dependent decrease in secreted IL-6 levels as measured via ELISA. In addition, myeloma cells pre-treated with the homogeranyl triazole compound displayed a decreased ability to adhere to HS-5 cells. In conclusion, these studies establish homogeranyl triazole bisphosphonate as the lead compound in a novel series of inhibitors against GGDPS and support the further development of GGDPS inhibitors as potential therapeutic agents for multiple myeloma. DisclosuresNo relevant conflicts of interest to declare.

Synthesis of isoprenoid bisphosphonate ethers through C-P bond formations: Potential inhibitors of geranylgeranyl diphosphate synthase.

A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme geranylgeranyl diphosphate synthase (GGDPS). Five of the new compounds show IC50 values of less than 1 μM against GGDPS with little to no activity against the related enzyme farnesyl diphosphate synthase (FDPS). The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 μM concentration.

Mutations in GERANYLGERANYL DIPHOSPHATE SYNTHASE 1 affect chloroplast development in Arabidopsis thaliana (Brassicaceae)

Within plastids, geranylgeranyl diphosphate synthase is a key enzyme in the isoprenoid biosynthetic pathway that catalyzes the formation of geranylgeranyl diphosphate, a precursor molecule to several biochemical pathways including those that lead into the biosynthesis of carotenoids and abscisic acid, prenyllipids such as the chlorophylls, and diterpenes such as gibberellic acid. • We have identified mutants in the GERANYLGERANYL DIPHOSPHATE SYNTHASE 1 (GGPS1) gene, which encodes the major plastid-localized enzyme geranylgeranyl diphosphate synthase in Arabidopsis thaliana. • Two T-DNA insertion mutant alleles (ggps1-2 and ggps1-3) were found to result in seedling-lethal albino and embryo-lethal phenotypes, respectively, indicating that GGPS1 is an essential gene. We also identified a temperature-sensitive leaf variegation mutant (ggps1-1) in A. thaliana that is caused by a point mutation. Total chlorophyll and carotenoid levels were reduced in ggps1-1 white tissues as compared with green tissues. Phenotypes typically associated with a reduction in gibberellic acid were not seen, suggesting that gibberellic acid biosynthesis is not noticeably altered in the mutant. In contrast to other variegated mutants, the ggps1-1 green sector photosynthetic rate was not elevated relative to wild-type tissues. Chloroplast development in green sectors of variegated leaves appeared normal, whereas cells in white sectors contained abnormal plastids with numerous electron translucent bodies and poorly developed internal membranes. • Our results indicate that GGPS1 is a key gene in the chlorophyll biosynthetic pathway.

Characterization and subcellular localization of geranylgeranyl diphosphate synthase from Catharanthus roseus

The enzyme geranylgeranyl diphosphate synthase (GGPS: EC 2.5.1.1, EC 2.5.1.10, EC 2.5.1.29) catalyses the formation of geranylgeranyl diphosphate (GGPP) from isopentenyl diphosphate and dimethylallyl diphosphate via three successive condensation reactions. A full-length nucleotide sequence of GGPS (named CrGGPS) was cloned from the medicinal plant Catharanthus roseus. The deduced polypeptide has 383 amino acids with a calculated mass of 41.6 kDa and possesses prenyltransferase signatures characteristic of plant type II GGPS. The enzyme was characterized by functional complementation in carotenoid accumulating strains of Escherichia coli. When cultures of Catharanthus cell lines were treated with methyljasmonate, no specific increase in transcript levels were observed. In plants, GGPS are encoded by a small multigene family and the isoforms have been shown to be localized in three different subcellular compartments: chloroplast, endoplasmic reticulum and mitochondria. We investigated the subcellular distribution of CrGGPS through transient transformations of C. roseus cells with a yellow fluorescent protein-fused construct. Our results clearly indicate that CrGGPS is located to plastids within stroma and stromules.

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.