Geranylgeranyl Diphosphate Synthase Inhibitors Research Articles

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.

Geranylgeranyl diphosphate synthase: Role in human health, disease and potential therapeutic target.

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthesis pathway, is responsible for the production of geranylgeranyl pyrophosphate (GGPP). GGPP serves as a substrate for the post-translational modification (geranylgeranylation) of proteins, including those belonging to the Ras superfamily of small GTPases. These proteins play key roles in signalling pathways, cytoskeletal regulation and intracellular transport, and in the absence of the prenylation modification, cannot properly localise and function. Aberrant expression of GGDPS has been implicated in various human pathologies, including liver disease, type 2 diabetes, pulmonary disease and malignancy. Thus, this enzyme is of particular interest from a therapeutic perspective. Here, we review the physiological function of GGDPS as well as its role in pathophysiological processes. We discuss the current GGDPS inhibitors under development and the therapeutic implications of targeting this enzyme.

Targeting the Isoprenoid Biosynthetic Pathway in Multiple Myeloma.

Multiple myeloma (MM) is a plasma cell malignancy for which there is currently no cure. While treatment options for MM have expanded over the last two decades, all patients will eventually become resistant to current therapies. Thus, there is an urgent need for novel therapeutic strategies to treat MM. The isoprenoid biosynthetic pathway (IBP) is responsible for the post-translational modification of proteins belonging to the Ras small GTPase superfamily, such as Ras, Rho and Rab family members. Given the important roles these GTPase proteins play in various cellular processes, there is significant interest in the development of inhibitors that disturb their prenylation and consequently their activity in MM cells. Numerous preclinical studies have demonstrated that IBP inhibitors have anti-MM effects, including the induction of apoptosis in MM cells and inhibition of osteoclast activity. Some IBP inhibitors have made their way into the clinic. For instance, nitrogenous bisphosphonates are routinely prescribed for the management MM bone disease. Other IBP inhibitors, including statins and farnesyltransferase inhibitors, have been evaluated in clinical trials for MM, while there is substantial preclinical investigation into geranylgeranyl diphosphate synthase inhibitors. Here we discuss recent advances in the development of IBP inhibitors, assess their mechanism of action and evaluate their potential as anti-MM agents.

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.

Geranylgeranyl diphosphate synthase inhibitor and proteasome inhibitor combination therapy in multiple myeloma

BackgroundMultiple myeloma (MM) remains an incurable malignancy, despite the advent of therapies such as proteosome inhibitors (PIs) that disrupt protein homeostasis and induce ER stress. We have pursued inhibition of geranylgeranyl diphosphate synthase (GGDPS) as a novel mechanism by which to target protein homeostasis in MM cells. GGDPS inhibitors (GGSI) disrupt Rab geranylgeranylation, which in turn results in perturbation of Rab-mediated protein trafficking, leading to accumulation of intracellular monoclonal protein, induction of ER stress and apoptosis. Our lead GGSI, RAM2061, has demonstrated favorable pharmacokinetic properties and in vivo efficacy. Here we sought to evaluate if combination therapy with GGSI and PI would result in enhanced disruption of the unfolded protein response (UPR) and increase anti-MM efficacy.MethodsMTT assays were conducted to evaluate the cytotoxic effects of combining RAM2061 with bortezomib in human MM cells. The effects of RAM2061 and/or PI (bortezomib or carfilzomib) on markers of UPR and apoptosis were evaluated by a combination of immunoblot (ATF4, IRE1, p-eIF2a, cleaved caspases and PARP), RT-PCR (ATF4, ATF6, CHOP, PERK, IRE1) and flow cytometry (Annexin-V). Induction of immunogenic cell death (ICD) was assessed by immunoblot (HMGB1 release) and flow cytometry (calreticulin translocation). Cell assays were performed using both concurrent and sequential incubation with PIs. To evaluate the in vivo activity of GGSI/PI, a flank xenograft using MM.1S cells was performed.ResultsIsobologram analysis of cytotoxicity data revealed that sequential treatment of bortezomib with RAM2061 has a synergistic effect in MM cells, while concurrent treatment was primarily additive or mildly antagonistic. The effect of PIs on augmenting RAM2061-induced upregulation of UPR and apoptotic markers was dependent on timing of the PI exposure. Combination treatment with RAM2061 and bortezomib enhanced activation of ICD pathway markers. Lastly, combination treatment slowed MM tumor growth and lengthened survival in a MM xenograft model without evidence of off-target toxicity.ConclusionWe demonstrate that GGSI/PI treatment can potentiate activation of the UPR and apoptotic pathway, as well as induce upregulation of markers associated with the ICD pathway. Collectively, these findings lay the groundwork for future clinical studies evaluating combination GGSI and PI therapy in patients with MM.

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.

Preclinical Investigation of a Potent Geranylgeranyl Diphosphate Synthase Inhibitor

The isoprenoid biosynthesis pathway (IBP) is responsible for the synthesis of isoprenoids used for the prenylation of proteins belonging to the Ras small GTPase superfamily. Geranylgeranyl diphosphate synthase (GGDPS) is the enzyme in the IBP pathway that catalyzes the synthesis of the 20-carbon isoprenoid GGDP. The enzymes geranylgeranyl transferase (GGTase) I and II utilize GGDP as the isoprenoid substrate for their geranylgeranylation reactions. Rab proteins mediate vesicle trafficking within the cell and their activity is dependent on geranylgeranylation. Our prior work has demonstrated that agents that disrupt Rab geranylgeranylation disrupt monoclonal protein trafficking in myeloma cells, resulting in induction of the unfolded protein response (UPR) pathway and apoptosis. Because myeloma cells express near maximal levels of UPR-associated proteins to aid in the continuous production of monoclonal proteins, they are particularly sensitive to activation of the pro-apoptotic arm of the UPR. We are therefore pursuing the development of GGDPS inhibitors as a novel therapeutic strategy for myeloma. Our lead compound VSW1198 (Figure) is the most potent GGDPS inhibitor reported to date with an IC50 of 45nM against purified enzyme, high specificity for GGDPS over the related enzyme farnesyl diphosphate synthase, and measurable cellular activity at concentrations as low as 30 nM (15 ng/mL). Due to its potent activity against myeloma cells in vitro, we were interested in evaluating the metabolic stability, pharmacokinetic (PK) profile, distribution pattern, and toxicology profile of VSW1198 in preparation for in vivo efficacy studies. Single dose testing via IV administration in CD-1 mice revealed a maximum tolerated dose of 0.5 mg/kg. Doses > 1mg/kg resulted in liver toxicity that peaked around 6-7 days post-injection. Histopathological examination of the livers demonstrated centrilobular hepatocyte necrosis and apoptosis with invasion of inflammatory cells and panlobular hepatocyte swelling. Analysis of blood samples showed elevated transaminase levels without effects on bilirubin, alkaline phosphatase, lactic dehydrogenase, or albumin, consistent with the observed pattern of hepatic injury. A control compound, RAM3059, also was tested in vivo . This compound has a similar isoprenoid triazole bisphosphonate structure (Figure) but is 380-fold less potent than VSW1198 as a GGDPS inhibitor. Mice injected with RAM3059 (1.6 mg/kg IV) were grossly and histologically normal with no signs of liver toxicity, suggesting liver injury is not due to nonspecific toxicity from the triazole bisphosphonate structure. No effects on kidney function, calcium, creatine kinase, or blood counts were observed. Histopathological examination of the brains and kidneys from the treated mice revealed no abnormalities. A PK and tissue distribution study included 11 blood samples obtained over a period from 5 minutes to 7 days post-injection of VSW1198 (0.5 mg/kg IV, n=5 per time point). Tissue samples (liver, kidney, spleen, lung, heart, brain, and bone marrow) were collected at 2, 8, 24, 48, and 72 hours post-injection (n=5 per time point). VSW1198 was extracted from plasma and tissue samples, derivatized by reaction with diazomethane, and analyzed via LC-MS/MS. The maximum concentration (C0 ) is 611.0 (+ 183.3) ng/mL, the area under curve (AUC0-∞ ) is 2355.9 (+ 160.3) hr*ng/mL, and the plasma elimination half-life (t1/2 ) is 38.0 (± 10.9) hrs. Detectable concentrations of VSW1198 at 72 hours (5.0 + 1.0 ng/mL) indicate prolonged systemic exposure. Analysis of tissues samples is underway and will be reported. The metabolic stability of VSW1198 was assessed using human liver microsomes (HLM) and mouse S9 cells for phase I and phase II metabolism. In both the HLM and mouse S9 studies, VSW1198 showed complete stability while a positive control showed rapid metabolic degradation. In summary, these studies demonstrate the metabolic stability and systemic distribution of the potent GGDPS inhibitor VSW1198. Multi-dose testing and efficacy studies in a myeloma xenograft mouse model are currently underway to assess further the clinical potential of GGDPS inhibitor therapy. [Display omitted] DisclosuresHolstein:Celgene: Consultancy; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees.

In vivo evaluation of combination therapy targeting the isoprenoid biosynthetic pathway

Geranylgeranyl diphosphate synthase (GGDPS), an enzyme in the isoprenoid biosynthetic pathway (IBP), produces the isoprenoid (geranylgeranyl pyrophosphate, GGPP) used in protein geranylgeranylation reactions. Our prior studies utilizing triazole bisphosphonate-based GGDPS inhibitors (GGSIs) have revealed that these agents represent a novel strategy by which to induce cancer cell death, including multiple myeloma and pancreatic cancer. Statins inhibit the rate-limiting enzyme in the IBP and potentiate the effects of GGSIs in vitro. The in vivo effects of combination therapy with statins and GGSIs have not been determined. Here we evaluated the effects of combining VSW1198, a novel GGSI, with a statin (lovastatin or pravastatin) in CD-1 mice. Twice-weekly dosing with VSW1198 at the previously established maximally tolerated dose in combination with a statin led to hepatotoxicity, while once-weekly VSW1198-based combinations were feasible. No abnormalities in kidney, spleen, brain or skeletal muscle were observed with combination therapy. Combination therapy disrupted protein geranylgeranylation in vivo. Evaluation of hepatic isoprenoid levels revealed decreased GGPP levels in the single drug groups and undetectable GGPP levels in the combination groups. Additional studies with combinations using 50% dose-reductions of either VSW1198 or lovastatin revealed minimal hepatotoxicity with expected on-target effects of diminished GGPP levels and disruption of protein geranylgeranylation. Combination statin/GGSI therapy significantly slowed tumor growth in a myeloma xenograft model. Collectively, these studies are the first to demonstrate that combination IBP inhibitor therapy alters isoprenoid levels and disrupts protein geranylgeranylation in vivo as well as slows tumor growth in a myeloma xenograft model, thus providing the framework for future clinical exploration.

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.

Conditional loss of geranylgeranyl diphosphate synthase alleviates acute obstructive cholestatic liver injury by regulating hepatic bile acid metabolism.

Previous studies have suggested that metabolites in the mevalonate pathway are involved in hepatic bile acid metabolism, yet the details of this relationship remain unknown. In this study, we found that the hepatic farnesyl pyrophosphate (FPP) level and the ratio of FPP to geranylgeranyl pyrophosphate (GGPP) were increased in mice with acute obstructive cholestasis compared with mice that underwent a sham operation. In addition, the livers of the mice with acute obstructive cholestasis showed lower expression of geranylgeranyl diphosphate synthase (GGPPS), which synthesizes GGPP from FPP. When Ggps1 was conditionally deleted in the liver, amelioration of liver injury, as shown by downregulation of the hepatic inflammatory response and decreased hepatocellular apoptosis, was found after ligation of the common bile duct and cholecystectomy (BDLC). Subsequently, liquid chromatography/mass spectrometry analysis showed that knocking out Ggps1 decreased the levels of hepatic bile acids, including hydrophobic bile acids. Mechanistically, the disruption of Ggps1 increased the levels of hepatic FPP and its metabolite farnesol, thereby resulting in farnesoid X receptor (FXR) activation, which modulated hepatic bile acid metabolism and reduced hepatic bile acids. It was consistently indicated that digeranyl bisphosphonate, a specific inhibitor of GGPPS, and GW4064, an agonist of FXR, could also alleviate acute obstructive cholestatic liver injury invivo. In general, GGPPS is critical for modulating acute obstructive cholestatic liver injury, and the inhibition of GGPPS ameliorates acute obstructive cholestatic liver injury by decreasing hepatic bile acids, which is possibly achieved through the activation of FXR-induced bile acid metabolism.

Geranylgeranyl Diphosphate Synthase Inhibitor and Proteasome Inhibitor Combination Therapy in Multiple Myeloma

Introduction: Multiple myeloma (MM) is characterized by proliferation of malignant plasma cells that produce monoclonal protein (MP). MM cells, compared with non-secretory cells, have a lower threshold for induction of the pro-apoptotic components of the unfolded protein response pathway (UPR) as a consequence of near-maximal expression of the protective UPR elements. Proteasome inhibitors (PIs) are widely used in the treatment of MM and have been shown to disrupt the UPR. As an alternative strategy by which to target protein homeostasis in MM, we have focused on the development of geranylgeranyl diphosphate synthase (GGDPS) inhibitors. Inhibition of GGDPS results in disruption of Rab protein geranylgeranylation, which in turn results in disruption of MP trafficking, leading to accumulation of intracellular MP, induction of the UPR and apoptosis. We have previously reported preclinical studies with our lead GGDPS inhibitor (GGDPSi), RAM2061, which demonstrated the agent's metabolic stability, prolonged half-life (plasma elimination half-life of 29.2 (+6.0) hours (hrs)), systemic distribution, in vivo disruption of geranylgeranylation and anti-tumor efficacy in a mouse MM xenograft model (Haney et al., ASH 2018, abstract 215). We hypothesized that the combination of GGDPSi and PI therapy would result in enhanced disruption of the UPR and increase anti-MM efficacy. Methods: MTT assays were conducted to evaluate the cytotoxic effects of combining RAM2061 with the PI bortezomib (Bor) in human MM cell lines (RPMI-8226, MM.1S) and isobologram analysis was performed. The effects of RAM2061 and/or Bor on markers of the UPR were evaluated via qRT-PCR (ATF4, ATF6, CHOP, PERK, IRE1) and immunoblot analysis (ATF4, IRE1, phosphorylated eIF2α). Apoptotic markers (cleaved caspases 3, 8 and 9 and cleaved PARP) were evaluated by immunoblot analysis. Cell assays were performed using both concurrent (both drugs added at the start of the 48 hr incubation period) and RAM2061 pre-treatment (RAM2061 added at the start, Bor added after 24 hrs) approaches. To evaluate the in vivo activity of the novel combination therapy, a flank xenograft model was used. NOD/SCID mice were subcutaneously inoculated in the flank with MM.1S cells. Once tumors became palpable, mice were randomly divided into four treatment groups (n=8 per group): control (PBS), RAM2061 (0.08 mg/kg IV two times/week (wk)), Bor (0.3 mg/kg SQ two times/wk) and RAM2061/Bor combination. Tumor volume (TV) and animal body weight were recorded three times/wk. Mice were euthanized when tumors reached 2000 mm3. Blood samples from day 9 post-initiation of treatment were analyzed via ELISA for human λ light chain. Blood samples collected at time of euthanasia were analyzed for hematologic, renal and hepatic parameters. Results: Isobologram analysis revealed a primarily additive effect with concurrent GGDPSi/PI incubations and primarily synergistic effect with the RAM2061 pre-treatment approach. The effect of Bor on augmenting RAM2061-induced upregulation of UPR and apoptotic markers was both concentration- and time-dependent. While there was in general an enhanced induction of apoptosis with the combination, Bor partially abrogated the RAM2061-induced upregulation of UPR markers at the tested time points. Studies are ongoing to determine whether this effect is associated with an accelerated time course of UPR activation in the presence of Bor. Consistent with our previous in vivo studies, the primary toxicity associated with RAM2061 treatment was hepatic transaminase elevation. No additional toxicity was observed when Bor was combined with RAM2061 based on animal weight, hematological, renal or hepatic parameters. Tumor growth over time was significantly decreased in the RAM2061 (p=0.0009) and RAM2061/Bor (p=0.0002) treatment groups compared to control animals and this corresponded to prolonged time to sacrifice (p=0.09 for RAM2061 group and p=0.003 for RAM2061/Bor group). Consistent with TV, mean plasma human λ light chain levels were decreased in the RAM2061 and RAM2061/Bor treatment groups compared to the control mice (p<0.05). Conclusion: These studies support further investigation into the therapeutic potential of GGDPSi/PI-based combination therapy for MM. Further studies are needed to dissect the mechanisms underlying the interactions between the two classes of drugs on the UPR and apoptotic pathways. Disclosures Holstein: Takeda: Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; Celgene: Consultancy; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Sorrento: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees.

Regulation of the Notch-ATM-abl axis by geranylgeranyl diphosphate synthase inhibition

Notch proteins drive oncogenesis of many cancers, most prominently T-cell acute lymphoblastic leukemia (T-ALL). Because geranylgeranylated Rab proteins regulate Notch processing, we hypothesized that inhibition of geranylgeranyl diphosphate synthase (GGDPS) would impair Notch processing and reduce viability of T-ALL cells that express Notch. Here, we show that GGDPS inhibition reduces Notch1 expression and impairs the proliferation of T-ALL cells. GGDPS inhibition also reduces Rab7 membrane association and depletes Notch1 mRNA. GGDPS inhibition increases phosphorylation of histone H2A.X, and inhibitors of ataxia telangiectasia-mutated kinase (ATM) mitigate GGDPS inhibitor-induced apoptosis. GGDPS inhibition also influences c-abl activity downstream of caspases, and inhibitors of these enzymes prevent GGDPS inhibitor-induced apoptosis. Surprisingly, induction of apoptosis by GGDPS inhibition is reduced by co-treatment with γ-secretase inhibitors. While inhibitors of γ-secretase deplete one specific form of the Notch1 intracellular domain (NICD), they also increase Notch1 mRNA expression and increase alternate forms of Notch1 protein expression in cells treated with a GGDPS inhibitor. Furthermore, inhibitors of γ-secretase and ATM increase Notch1 mRNA stability independent of GGDPS inhibition. These results provide a model by which T-ALL cells use Notch1 to avoid DNA-damage-induced apoptosis, and can be overcome by inhibition of GGDPS through effects on Notch1 expression and its subsequent response.

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.

Metal Coordination Is Crucial for Geranylgeranyl Diphosphate Synthase-Bisphosphonate Interactions: A Crystallographic and Computational Analysis.

Geranylgeranyl diphosphate synthase (GGPPS) is a central metalloenzyme in the mevalonate pathway, crucial for the prenylation of small GTPases. As small GTPases are pivotal for cellular survival, GGPPS was highlighted as a potential target for treating human diseases, including solid and hematologic malignancies and parasitic infections. Most available GGPPS inhibitors are bisphosphonates, but the clinically available compounds demonstrate poor pharmacokinetic properties. Although the design of novel bisphosphonates with improved physicochemical properties is highly desirable, the structure of wild-type human GGPPS (hGGPPS) bound to a bisphosphonate has not been resolved. Moreover, various metal-bisphosphonate-binding stoichiometries were previously reported in structures of yeast GGPPS (yGGPPS), hampering computational drug design with metal-binding pharmacophores (MBP). In this study, we report the 2.2 Å crystal structure of hGGPPS in complex with ibandronate, clearly depicting the involvement of three Mg2+ ions in bisphosphonate-protein interactions. Using drug-binding assays and computational docking, we show that the assignment of three Mg2+ ions to the binding site of both hGGPPS and yGGPPS greatly improves the correlation between calculated binding energies and experimentally measured affinities. This work provides a structural basis for future rational design of additional MBP-harboring drugs targeting hGGPPS. SIGNIFICANCE STATEMENT: Bisphosphonates are inhibitors of geranylgeranyl diphosphate synthase (GGPPS), a metalloenzyme crucial for cell survival. Bisphosphonate binding depends on coordination by Mg2+ ions, but various Mg2+-bisphosphonate-binding stoichiometries were previously reported. In this study, we show that three Mg2+ ions are vital for drug binding and provide a structural basis for future computational design of GGPPS 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.

Inhibition of geranylgeranyl diphosphate synthase is a novel therapeutic strategy for pancreatic ductal adenocarcinoma.

Rab proteins play an essential role in regulating intracellular membrane trafficking processes. Rab activity is dependent upon geranylgeranylation, a post-translational modification that involves the addition of 20-carbon isoprenoid chains via the enzyme geranylgeranyl transferase (GGTase) II. We have focused on the development of inhibitors against geranylgeranyl diphosphate synthase (GGDPS), which generates the isoprenoid donor (GGPP), as anti-Rab agents. Pancreatic ductal adenocarcinoma (PDAC) is characterized by abnormal mucin production and these mucins play important roles in tumor development, metastasis and chemo-resistance. We hypothesized that GGDPS inhibitor (GGDPSi) treatment would induce PDAC cell death by disrupting mucin trafficking, thereby inducing the unfolded protein response pathway (UPR) and apoptosis. To this end, we evaluated the effects of RAM2061, a potent GGDPSi, against PDAC. Our studies revealed that GGDPSi treatment activates the UPR and triggers apoptosis in a variety of human and mouse PDAC cell lines. Furthermore, GGDPSi treatment was found to disrupt the intracellular trafficking of key mucins such as MUC1. These effects could be recapitulated by incubation with a specific GGTase II inhibitor, but not a GGTase I inhibitor, consistent with the effect being dependent on disruption of Rab-mediated activities. In addition, siRNA-mediated knockdown of GGDPS induces upregulation of UPR markers and disrupts MUC1 trafficking in PDAC cells. Experiments in two mouse models of PDAC demonstrated that GGDPSi treatment significantly slows tumor growth. Collectively, these data support further development of GGDPSi therapy as a novel strategy for the treatment of PDAC.

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.

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.

Preclinical investigation of a potent geranylgeranyl diphosphate synthase inhibitor.

Geranylgeranyl diphosphate synthase (GGDPS) is the enzyme in the isoprenoid biosynthesis pathway that catalyzes the synthesis of the 20-carbon isoprenoid GGPP, which serves as the isoprenoid donor for protein geranylgeranylation reactions. Rab proteins mediate vesicle trafficking within the cell and their activity is dependent on geranylgeranylation. Our prior work has demonstrated that agents that disrupt Rab geranylgeranylation disrupt monoclonal protein trafficking in myeloma cells, resulting in induction of the unfolded protein response pathway and apoptosis. VSW1198 is a potent GGDPS inhibitor with measurable cellular activity at concentrations as low as 30nM. Due to its potent activity against myeloma cells in vitro, we were interested in evaluating the toxicology profile, pharmacokinetic (PK) profile, tissue distribution pattern and metabolic stability of VSW1198 in preparation for in vivo efficacy studies. Single dose testing via IV administration in CD-1 mice revealed a maximum tolerated dose of 0.5mg/kg. Doses ≥1mg/kg resulted in liver toxicity that peaked around 6-7days post-injection. Disruption of protein geranylgeranylation following repeat dosing of VSW1198 was confirmed via immunoblot analysis of unmodified Rap1a in multiple organs. The PK studies revealed a half-life of 47.7 ± 7.4h. VSW1198 was present in all tested tissues with the highest levels in the liver. In both human liver microsomes and mouse S9 studies VSW1198 showed complete stability, suggesting no phase I or phase II metabolism. In summary, these studies demonstrate systemic distribution, on-target disruption of protein geranylgeranylation, and metabolic stability of a potent GGDPS inhibitor VSW1198 and form the basis for future efficacy studies in mouse models of myeloma.

Quantitative determination of a potent geranylgeranyl diphosphate synthase inhibitor using LC–MS/MS: Derivatization and application

An isomeric mixture of homogeranyl/homoneryl triazole bisphosphonates (VSW1198) has previously been shown to be a potent inhibitor of geranylgeranyl diphosphate (GGDP) synthase (GGDPS) and of therapeutic interest for the treatment of multiple myeloma. We have developed and validated a selective and sensitive liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantitation of both the E- and Z- isomers of VSW1198 in cell culture media, mouse plasma and tissues. VSW1198 and internal standard are extracted from the bio-matrices by solid-phase extraction, followed by derivatization using trimethylsilyldiazomethane. The chromatographic separation of analytes was achieved on a Phenomenex Gemini NX column (150 mm * 2.0 mm, 5 μ) with gradient elution using 0.1% acetic acid and methanol/acetonitrile (1:1) as the mobile phase at a flow rate of 0.2 mL/min. Derivatized analytes were ionized with an electrospray ionization source in positive multiple reaction monitoring (MRM) mode and quantitated using MS/MS. The MS/MS response was linear over the concentration range from 0.38–1500 and 0.13–500 ng/mL for the E- and Z-isomers, respectively. The within- and between-day precision (relative standard deviation, % RSD) and accuracy were within the acceptable limits per FDA guidelines. The validated method was used for quantitative determination of the compounds in preclinical studies focused on the development of VSW1198 as a novel anti-cancer agent.