Inhibition Of Farnesyl Diphosphate Synthase Research Articles

Structural Insights into the Substrate Binding of Farnesyl Diphosphate Synthase FPPS1 from Silkworm, Bombyx mori.

Farnesyl diphosphate synthase (FPPS) is an important enzyme involved in the juvenile hormone (JH) biosynthesis pathway. Herein, we report the crystal structure of a type-I Lepidopteran FPPS from Bombyx mori (BmFPPS1) at 2.80 Å resolution. BmFPPS1 adopts an α-helix structure with a deep cavity at the center of the overall structure. Computational simulations combined with biochemical analysis allowed us to define the binding mode of BmFPPS1 to its substrates. Structural comparison revealed that BmFPPS1 adopts a structural pattern similar to that of type-II FPPS but exhibits a distinct substrate-binding site. These findings provide a structural basis for understanding substrate preferences and designing FPPS inhibitors. Furthermore, the expression profiles and RNA interference of BmFPPSs indicated that they play critical roles in the JH biosynthesis and larval-pupal metamorphosis. These findings enhance our understanding of the structural features of type-I Lepidopteran FPPS while providing direct evidence for the physiological role of BmFPPSs in silkworm development.

CD133+ endothelial-like stem cells restore neovascularization and promote longevity in progeroid and naturally aged mice

The stem cell theory of aging dictates that a decline in the number and/or function of stem cells causes tissue degeneration and aging; however, it still lacks unequivocal experimental support. Here, using lineage tracing and single-cell transcriptomics, we identify a population of CD133+ bone marrow-derived endothelial-like cells (ELCs) as potential endothelial progenitor cells, which contribute to tubular structures in vitro and neovascularization in vivo. We demonstrate that supplementation with wild-type and young ELCs respectively restores neovascularization and extends lifespan in progeric and naturally aged mice. Mechanistically, we identify an upregulation of farnesyl diphosphate synthase (FDPS) in aged CD133+ ELCs—a key enzyme in isoprenoid biosynthesis. Overexpression of FDPS compromises the neovascularization capacity of CD133+ ELCs, whereas FDPS inhibition by pamidronate enhances neovascularization, improves health measures and extends lifespan in aged mice. These findings highlight stem cell-based strategies for the treatment of progeria and age-related pathologies.

B7H3-targeting chimeric antigen receptor modification enhances antitumor effect of Vγ9Vδ2 T cells in glioblastoma

BackgroundGlioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. This study investigates the therapeutic potential of human Vγ9Vδ2 T cells in GBM treatment. The sensitivity of different glioma specimens to Vγ9Vδ2 T cell-mediated cytotoxicity is assessed using a patient-derived tumor cell clusters (PTCs) model.MethodsThe study evaluates the anti-tumor effect of Vγ9Vδ2 T cells in 26 glioma cases through the PTCs model. Protein expression of BTN2A1 and BTN3A1, along with gene expression related to lipid metabolism and glioma inflammatory response pathways, is analyzed in matched tumor tissue samples. Additionally, the study explores two strategies to re-sensitize tumors in the weak anti-tumor effect (WAT) group: utilizing a BTN3A1 agonistic antibody or employing bisphosphonates to inhibit farnesyl diphosphate synthase (FPPS). Furthermore, the study investigates the efficacy of genetically engineered Vγ9Vδ2 T cells expressing Car-B7H3 in targeting diverse GBM specimens.ResultsThe results demonstrate that Vγ9Vδ2 T cells display a stronger anti-tumor effect (SAT) in six glioma cases, while showing a weaker effect (WAT) in twenty cases. The SAT group exhibits elevated protein expression of BTN2A1 and BTN3A1, accompanied by differential gene expression related to lipid metabolism and glioma inflammatory response pathways. Importantly, the study reveals that the WAT group GBM can enhance Vγ9Vδ2 T cell-mediated killing sensitivity by incorporating either a BTN3A1 agonistic antibody or bisphosphonates. Both approaches support TCR-BTN mediated tumor recognition, which is distinct from the conventional MHC-peptide recognition by αβ T cells. Furthermore, the study explores an alternative strategy by genetically engineering Vγ9Vδ2 T cells with Car-B7H3, and both non-engineered and Car-B7H3 Vγ9Vδ2 T cells demonstrate promising efficacy in vivo, underscoring the versatile potential of Vγ9Vδ2 T cells for GBM treatment.ConclusionsVγ9Vδ2 T cells demonstrate a robust anti-tumor effect in some glioma cases, while weaker in others. Elevated BTN2A1 and BTN3A1 expression correlates with improved response. WAT group tumors can be sensitized using a BTN3A1 agonistic antibody or bisphosphonates. Genetically engineered Vγ9Vδ2 T cells, i.e., Car-B7H3, show promising efficacy. These results together highlight the versatility of Vγ9Vδ2 T cells for GBM treatment.

Inhibitors of Farnesyl Diphosphate Synthase and Squalene Synthase: Potential Source for Anti-Trypanosomatidae Drug Discovery

Trypanosomatids are mainly responsible for leishmaniasis, sleeping sickness, and Chagas disease, which are the most challenging among the neglected tropical diseases due to the problem of drug resistance. Although problems of target deconvolution and polypharmacology are encountered, a target-based approach is a rational method for screening drug candidates targeting a biomolecule that causes infections. The present study aims to summarize the latest information regarding potential inhibitors of squalene synthase and farnesyl phosphate synthase with anti-Trypanosomatidae activity. The information was obtained by referencing textbooks and major scientific databases from their inception until April 2023. Based on in vitro experiments, more than seventy compounds were reported to inhibit squalene synthase and farnesyl diphosphate synthase. Among these compounds, more than 30 were found to be active in vitro against Trypanosomatidae, inferring that these compounds can be used as scaffolds to develop new drugs against trypanosomatid-related infections. Overall, natural and synthetic products can inhibit enzymes that are crucial for the survival and virulence of trypanosomatids. Moreover, in vitro experiments have confirmed the activity of more than half of these inhibitors using cell-based assays. Nevertheless, additional studies on the cytotoxicity, pharmacokinetics, and lead optimization of potent anti-Trypanosomatid compounds should be investigated.

Regulation of hepatic coenzyme Q biosynthesis by dietary omega-3 polyunsaturated fatty acids

Dietary fats are important for human health, yet it is not fully understood how different fats affect various health problems. Although polyunsaturated fatty acids (PUFAs) are generally considered as highly oxidizable, those of the n-3 series can ameliorate the risk of many age-related disorders. Coenzyme Q (CoQ) is both an essential component of the mitochondrial electron transport chain and the only lipid-soluble antioxidant that animal cells can synthesize. Previous work has documented the protective antioxidant properties of CoQ against the autoxidation products of PUFAs. Here, we have explored in vitro and in vivo models to better understand the regulation of CoQ biosynthesis by dietary fats. In mouse liver, PUFAs increased CoQ content, and PUFAs of the n-3 series increased preferentially CoQ10. This response was recapitulated in hepatic cells cultured in the presence of lipid emulsions, where we additionally demonstrated a role for n-3 PUFAs as regulators of CoQ biosynthesis via the upregulation of several COQ proteins and farnesyl pyrophosphate levels. In both models, n-3 PUFAs altered the mitochondrial network without changing the overall mitochondrial mass. Furthermore, in cellular systems, n-3 PUFAs favored the synthesis of CoQ10 over CoQ9, thus altering the ratio between CoQ isoforms through a mechanism that involved downregulation of farnesyl diphosphate synthase activity. This effect was recapitulated by both siRNA silencing and by pharmacological inhibition of farnesyl diphosphate synthase with zoledronic acid. We highlight here the ability of n-3 PUFAs to regulate CoQ biosynthesis, CoQ content, and the ratio between its isoforms, which might be relevant to better understand the health benefits associated with this type of fat. Additionally, we identify for the first time zoledronic acid as a drug that inhibits CoQ biosynthesis, which must be also considered with respect to its biological effects on patients.

Abstract 46: A new class of precision therapeutics that inhibit prostate cancer mediated bone destruction

Abstract The destructive nature of bone metastases dominates the clinical management of advanced prostate cancer (PCa) and is a common cause of morbidity and mortality. Our current ability to therapeutically target this process is limited. Here we describe the synthesis and functional characterization of the first-in-class Dual Acting Bone Defending agents (DABDs), which couple a small molecule therapeutic that inhibits cell movement to bone trophic bisphosphonates. Upfront in-vitro screens assessed DABD efficacy at inhibiting PCa cell migration and invasion and osteoclast mediated bone destruction. Systemic models of PCa metastasis, inclusive of intracardiac (IC) and intratibial (IT) injection models, were deployed to characterize tolerance and efficacy in-vivo. Finally, in-vitro characterization of DABD molecular mechanism of action was performed, as guided by the existing knowledge base related to the parental compounds. Results demonstrated that DABDs inhibit PCa cell migration and invasion, chemically bind hydroxyappetite, and inhibit osteoclast mediated bone destruction in vitro. Following IC injection of PCa cells, a model where cells are placed into circulation, metastasize to bone, grow and induce bone destruction, emulating the clinical scenario in humans, DABD treatment inhibited circulating PCa cells from forming new bone metastasis and prolonged life in a dose-dependent fashion. In the IT mouse models of established metastatic lesions, DABDs induce reversion of PCa cell growth and disrupt further bone destruction. DABDs at doses 1000 fold higher than those associated with efficacy are well tolerated. At the molecular level, DABDs retain the molecular mechanisms of action of their constituent chemicals. They inhibit farnesyl diphosphate synthase (FDPS), in the mevalonate pathway, leading to decreased prenylation of small GTPases, including the Ras related protein, Rap-1A, as well as inhibit Raf1 activation, inducing apoptosis and impaired cell movement, respectively. DABDs are a new class of agents that inhibit formation of new bone metastasis, revert growth of established metastasis, prolong life, and have a high potential for improving clinical outcome in humans. Citation Format: Ryan R. Gordon, Abhinandan Pattanayak, Wenqi Li, Raymond Bergan. A new class of precision therapeutics that inhibit prostate cancer mediated bone destruction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 46.

Structure-Function Relationships of Novel Benzimidazole-Based Inhibitors of the Isoprenoid Biosynthetic Pathway in Myeloma Cells

Malignant plasma cells are characterized by the production of monoclonal protein (MP). Our work has demonstrated that disrupting intracellular MP trafficking by targeting the geranylgeranylation of Rab GTPases results in endoplasmic reticulum stress, induction of the unfolded protein response pathway and apoptosis of myeloma cells. Rab proteins are geranylgeranylated by the enzyme geranylgeranyl transferase II (GGTase II) which uses geranylgeranyl diphosphate as the isoprenoid substrate. We have pursued the development of specific GGTase II and geranylgeranyl diphosphate synthase (GGDPS) inhibitors as potential anti-myeloma agents. A family of compounds incorporating a benzimidazole heterocycle and either a carboxyphosphonate or bisphosphonate was prepared by modification of the appropriate heterocycles. The core benzimidazole phosphonate structure was modified with substituents at either the alpha-carbon position or at the 5 and 6 positions on the benzimidazole (Figure). These compounds were subjected to in vitro enzyme assays for GGTase II and the related prenyltransferases farnesyl transferase (FTase) and GGTase I as well as the synthases farnesyl diphosphate synthase (FDPS) and GGDPS. Cellular disruption of protein prenylation was assessed via immunoblot analysis for Ras (a representative farnesylated protein) and for unmodified Rap1a (a representative geranylgeranylated protein) in human myeloma cells (RPMI-8226, MM.1S). The ability of the novel compounds to disrupt MP trafficking was determined by measuring changes in intracellular lambda light chain levels via ELISA. Confirmation of disruption of Rab geranylgeranylation was achieved by Triton X-114 lysis to obtain detergent (membrane) fractions which were subjected to Rab6 (a representative Rab protein) immunoblot analysis. These studies demonstrated that the nature of the phosphonate head group has a profound impact on enzyme specificity: the carboxyphosphonates have specific activity as GGTase II inhibitors while the bisphosphonate analogues have specific activity as FDPS inhibitors with no cross-reactivity against any of the other tested enzymes. The most potent GGTase II and FDPS inhibitors were the unsubstituted analogues. The carboxyphosphonate 1 was found to have an IC50 of 230 μM against GGTase II and cellular activity starting at 2.5 mM. This represents a two-fold improvement in potency compared with the known GGTase II inhibitor 3-PEHPC. The bisphosphonate 5 has an IC50 of 24 nM against FDPS and cellular activity at ≥ 50 μM. The addition of a methyl group at the alpha-carbon position abrogated the activity of the carboxyphosphonate (2) as a GGTase II inhibitor and the activity of the bisphosphonate (6) as a FDPS inhibitor. The addition of substituents at the 5 and 6 positions (compounds 3, 4, 7 and 8)did not improve potency compared with the parent compounds. Co-incubation with low concentrations of the HMG-CoA reductase inhibitor lovastatin (0.1-1 μM), concentrations which on their own are not sufficient to disrupt protein prenylation, potentiated the cellular activity of the lead GGTase II and FDPS inhibitors. In conclusion, these benzimidazole phosphonates represent novel classes of GGTase II and FDPS inhibitors. These studies highlight the importance of the identity of the phosphonate head-group in dictating enzyme specificity. Further studies are underway to prepare additional benzimidazole analogues in order to improve their potency, advance the understanding of the structure-function relationship, and assess the potential of these compounds as anti-myeloma agents. [Display omitted] DisclosuresWiemer:Terpenoid Therapeutics, Inc: Other: Founder. Holstein:Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy.

Zoledronate repositioning as a potential trypanocidal drug. Trypanosoma cruzi HPRT an alternative target to be considered

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and affects 7 million people worldwide. Considering the side effects and drug resistance shown by current treatments, the development of new anti-Chagas therapies is an urgent need. T. cruzi hypoxanthine phosphoribosyltransferase (TcHPRT), the key enzyme of the purine salvage pathway, is essential for the survival of trypanosomatids. Previously, we assessed the inhibitory effect of different bisphosphonates (BPs), HPRT substrate analogues, on the activity of the isolated enzyme. BPs are used as a treatment for bone diseases and growth inhibition studies on T. cruzi have associated BPs action with the farnesyl diphosphate synthase inhibition. Here, we demonstrated significant growth inhibition of epimastigotes in the presence of BPs and a strong correlation with our previous results on the isolated TcHPRT, suggesting this enzyme as a possible and important target for these drugs. We also found that the parasites exhibited a delay at S phase in the presence of zoledronate pointing out enzymes involved in the cell cycle, such as TcHPRT, as intracellular targets. Moreover, we validated that micromolar concentrations of zoledronate are capable to interfere with the progression of cell infection by this parasite. Altogether, our findings allow us to propose the repositioning of zoledronate as a promising candidate against Chagas disease and TcHPRT as a new target for future rational design of antiparasitic drugs.

Zoledronic acid inhibits the growth of leukemic MLL-AF9 transformed hematopoietic cells

A leukemic in vitro model produced by transducing Cord Blood derived-hematopoietic CD34+ cells with the MLL-AF9 translocation resulting in the oncogenic fusion protein, is used to assess for sensitivity to Zoledronic acid. These cells are practically immortalized and are of myeloid origin. Proliferation, clonogenic and stromal co-culture assays showed that the MLL-AF9 cells were considerably more sensitive to Zoledronic acid than normal hematopoietic CD34+ cells or MS-5 stromal cells. The MLL-AF9 cells were notably more inhibited by Zoledronic acid when cultured as colonies in 3 dimensions, requiring cell-cell contacts compared to suspension expansion cultures. This is coherent with the mechanism of action of Zoledronic acid inhibiting farnesyl diphosphate synthase which results in a block in prenylation of GTPases such that their role in the membrane is compromised for cell-cell contacts. Zoledronic acid can be proposed to target the MLL-AF9 leukemic stem cells before they emerge from the hematopoietic niche, which being in proximity to bone osteoclasts where Zoledronic acid is sequestered can be predicted to result in sufficient levels to result in an anti-leukemic action.

ATRAID regulates the action of nitrogen-containing bisphosphonates on bone.

Nitrogen-containing bisphosphonates (N-BPs), such as alendronate, are the most widely prescribed medications for diseases involving bone, with nearly 200 million prescriptions written annually. Recently, widespread use of N-BPs has been challenged due to the risk of rare but traumatic side effects such as atypical femoral fracture (AFF) and osteonecrosis of the jaw (ONJ). N-BPs bind to and inhibit farnesyl diphosphate synthase, resulting in defects in protein prenylation. Yet, it remains poorly understood what other cellular factors might allow N-BPs to exert their pharmacological effects. Here, we performed genome-wide studies in cells and patients to identify the poorly characterized gene, ATRAID Loss of ATRAID function results in selective resistance to N-BP-mediated loss of cell viability and the prevention of alendronate-mediated inhibition of prenylation. ATRAID is required for alendronate inhibition of osteoclast function, and ATRAID-deficient mice have impaired therapeutic responses to alendronate in both postmenopausal and senile (old age) osteoporosis models. Last, we performed exome sequencing on patients taking N-BPs that suffered ONJ or an AFF. ATRAID is one of three genes that contain rare nonsynonymous coding variants in patients with ONJ or an AFF that is also differentially expressed in poor outcome groups of patients treated with N-BPs. We functionally validated this patient variation in ATRAID as conferring cellular hypersensitivity to N-BPs. Our work adds key insight into the mechanistic action of N-BPs and the processes that might underlie differential responsiveness to N-BPs in people.

A patent review of bisphosphonates in treating bone disease

ABSTRACTIntroduction: Bisphosphonates (BPs) are widely used to manage a variety of bone disorders, including osteoporosis, metastatic bone disease and myeloma bone disease. The nitrogen-containing BPs (NBPs) target osteoclast activity by disrupting protein prenylation via inhibition of farnesyl diphosphate synthase (FDPS).Areas covered: This review summarizes the recent advances in BPs with a focus on the latest patents (2015–2018). Patents involving novel BPs, new modes of BP delivery, as well as use of BPs to deliver other drugs to bone are discussed. A review of phosphonate-based drugs targeting geranylgeranyl diphosphate synthase (GGDPS) or geranylgeranyl transferase II (GGTase II) as alternative strategies to disrupt protein geranylgeranylation is provided.Expert opinion: While the NBPs remain the mainstay of treatment for most bone disorders, further understanding of their pharmacological properties could lead to further refinement of their chemical structures and optimization of efficacy and safety profiles. In addition, the development of NBP analogs or drug delivery mechanisms that allow for nonbone tissue exposure could allow for the use of these drugs as direct anticancer agents. The development of GGDPS and GGTase II inhibitors represents alternative heterocycle phosphonate-based strategies to disrupt protein geranylgeranylation and may have potential as anticancer agents and/or as bone-targeted therapies.

Farnesyl diphosphate synthase is important for the maintenance of glioblastoma stemness

Glioblastoma is a highly malignant tumor that easily acquires resistance to treatment. The stem-cell-like character (stemness) has been thought to be closely associated with the treatment resistance of glioblastoma cells. In this study, we determined that farnesyl diphosphate synthase (FDPS), a key enzyme in isoprenoid biosynthesis, plays an important role in maintaining glioblastoma stemness. A comparison of the mRNA expression in patient-derived glioblastoma sphere cells, which maintain stemness, and their differentiated counterparts, which lose stemness, via RNA sequencing showed that most of the altered genes were networked in the cholesterol biosynthesis pathway. We screened Federal Drug Administration (FDA)-approved drugs targeting specific enzymes in the cholesterol biosynthesis pathway for their ability to inhibit glioblastoma sphere formation. Inhibitors of FDPS, such as alendronate and zoledronate, significantly reduced the formation of glioblastoma spheres, and alendronate was effective at a lower molar concentration than zoledronate. Knockdown of FDPS using short hairpin RNA also completely inhibited the formation of secondary spheres. FDPS mRNA in patients with glioblastoma was associated with malignancy in three independent microarray data sets. RNA sequencing showed that alendronate treatment reduced the embryonic stem cell signature and activated development- and necrosis-related pathways in glioblastoma spheres. These results suggest that FDPS is important for the maintenance of glioblastoma stemness and that alendronate, a drug widely used to treat osteoporosis, can be repositioned to treat glioblastoma.

Targeting the phospholipase A2 receptor ameliorates premature aging phenotypes.

Hutchinson–Gilford progeria syndrome (HGPS) is a lethal premature aging that recapitulates many normal aging characteristics. This disorder is caused by mutation in the LMNA gene leading to the production of progerin which induces misshapen nuclei, cellular senescence, and aging. We previously showed that the phospholipase A2 receptor (PLA2R1) promotes senescence induced by replicative, oxidative, and oncogenic stress but its role during progerin‐induced senescence and in progeria is currently unknown. Here, we show that knockdown of PLA2R1 prevented senescence induced by progerin expression in human fibroblasts and markedly delayed senescence of HGPS patient‐derived fibroblasts. Whole‐body knockout of Pla2r1 in a mouse model of progeria decreased some premature aging phenotypes, such as rib fracture and decreased bone content, together with decreased senescence marker. Progerin‐expressing human fibroblasts exhibited a high frequency of misshapen nuclei and increased farnesyl diphosphate synthase (FDPS) expression compared to controls; knockdown of PLA2R1 reduced the frequency of misshapen nuclei and normalized FDPS expression. Pamidronate, a FDPS inhibitor, also reduced senescence and misshapen nuclei. Downstream of PLA2R1, we found that p53 mediated the progerin‐induced increase in FDPS expression and in misshapen nuclei. These results suggest that PLA2R1 mediates key premature aging phenotypes through a p53/FDPS pathway and might be a new therapeutic target.

Identifying Potential Therapeutics for Osteoporosis by Exploiting the Relationship between Mevalonate Pathway and Bone Metabolism.

Osteoporosis is a silent skeletal disease characterized by low bone mass and destruction of skeletal microarchitecture, leading to an increased fracture risk. This occurs due to an imbalance in bone remodelling, whereby the rate of bone resorption is greater than bone formation. Mevalonate pathway, previously known to involve in cholesterol synthesis, is an important regulatory pathway for bone remodelling. This review aimed to provide an overview of the relationship between mevalonate pathway and bone metabolism, as well as agents which act through this pathway to achieve their therapeutic potential. Mevalonate pathway produces farnesyl pyrophosphate and geranylgeranyl pyrophosphate essential in protein prenylation. An increase in protein prenylation favours bone resorption over bone formation. Non-nitrogen containing bisphosphonates inhibit farnesyl diphosphate synthase which produces farnesyl pyrophosphate. They are used as the first line therapy for osteoporosis. Statins, a well-known class of cholesterol-lowering agents, inhibit 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase, the rate-determining enzyme in the mevalonate pathway. It was shown to increase bone mineral density and prevent fracture in humans. Tocotrienol is a group of vitamin E commonly found in palm oil, rice bran and annatto bean. It causes degradation of HMG-CoA reductase. Many studies demonstrated that tocotrienol prevented bone loss in animal studies but its efficacy has not been tested in humans. Mevalonate pathway can be exploited to develop effective antiosteoporosis agents.

Gamma Delta T Cell Therapy for Cancer: It Is Good to be Local.

Human gamma delta T cells have extraordinary properties including the capacity for tumor cell killing. The major gamma delta T cell subset in human beings is designated Vγ9Vδ2 and is activated by intermediates of isoprenoid biosynthesis or aminobisphosphonate inhibitors of farnesyldiphosphate synthase. Activated cells are potent for killing a broad range of tumor cells and demonstrated the capacity for tumor reduction in murine xenotransplant tumor models. Translating these findings to the clinic produced promising initial results but greater potency is needed. Here, we review the literature on gamma delta T cells in cancer therapy with emphasis on the Vγ9Vδ2 T cell subset. Our goal was to examine obstacles preventing effective Vγ9Vδ2 T cell therapy and strategies for overcoming them. We focus on the potential for local activation of Vγ9Vδ2 T cells within the tumor environment to increase potency and achieve objective responses during cancer therapy. The gamma delta T cells and especially the Vγ9Vδ2 T cell subset, have the potential to overcome many problems in cancer therapy especially for tumors with no known treatment, lacking tumor-specific antigens for targeting by antibodies and CAR-T, or unresponsive to immune checkpoint inhibitors. Translation of amazing work from many laboratories studying gamma delta T cells is needed to fulfill the promise of effective and safe cancer immunotherapy.

Polyunsaturated fatty acids directly regulate coenzyme Q biosynthesis

Biological membranes adapt their phospholipid composition according to the major lipid source present in the diet. Different dietary sources may modify the lipid pattern and produce biochemical alterations in cells, especially in mitochondrial membranes. For example, sources of polyunsaturated fatty acids, such as soybean or fish oil, will generate membranes more susceptible to oxidative stress than will sources of saturated or monounsaturated fatty acids, such as animal fat or olive oil, respectively. Previous studies revealed that different dietary fats also influenced the mitochondrial levels of coenzyme Q (Q), a lipid present in all organisms. Apart from participating as an electron and proton carrier in the mitochondrial electron transport chain, Q serves numerous cellular functions including metabolism, antioxidant protection, and the regulation of signal transduction. We used a hepatocellular model of Hepa1‐6 cells treated with different lipid emulsions, and focused on the regulation of Q biosynthesis and the ultrastructure of the mitochondria. Our results indicate that treatment with unsaturated fatty acids increased Q levels, which corresponded to an increase in Q biosynthetic rate in the case of polyunsaturated fatty acids (PUFA) but not in the case of monounsaturated fatty acids (MUFA). Moreover, our results indicate that PUFA regulate the different Q isoforms, and promote the biosynthesis of Q10 over Q9 thus decreasing Q9/Q10 ratio. Since most of the cellular Q is located in mitochondria, the structure, number, size and distribution of this organelle greatly influences the overall content of Q in cells. We studied the ultrastructure and the abundance of mitochondria in order to evaluate whether the regulation of Q levels by fatty acids involved an alteration of mitochondrial ultrastructure and/or mitochondrial abundance. Electron microscopy micrographs of cells showed that cells treated with n‐3 PUFA showed significantly increased mitochondrial volume and the number of mitochondria per cell, explaining in part the increase of Q levels previously described. However, these alterations in the mitochondrial ultrastructure do not explain the alteration of the Q9/Q10 ratio. Further experiments focused on the mevalonate pathway showed that the alteration of the Q9/Q10 ratio can be explained by PUFA‐mediated inhibition of farnesyl diphosphate synthase, a key enzyme in this pathway. However, the observed increase of Q biosynthesis may implicate additional target(s). Immunostaining analyses reveal that PUFA exert an stimulatory effect on some of the COQ proteins involved in Q biosynthesis pathway. Further studies will be needed to fully understand the exact regulation that fatty acids exert on Q metabolism and mitochondrial ultrastructure.Support or Funding InformationBFU2015‐64630‐R and BFU2011‐23578 Projects from the Spanish Ministery of Economy. 1R01AG028125‐01A1 Project from NIH. FPU12/03398 Scholarship from the Spanish Ministery of Education, Culture and Sport.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Combining Vγ9Vδ2 T Cells with a Lipophilic Bisphosphonate Efficiently Kills Activated Hepatic Stellate Cells.

Activated hepatic stellate cells (aHSCs) are now established as a central driver of fibrosis in human liver injury. In the presence of chronic or repeated injury, fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) can occur, so there is interest in down-regulating aHSCs activity in order to treat these diseases. Here, we report that Vγ9Vδ2 T cells are reduced in patients with liver cirrhosis, stimulating us to investigate possible interactions between Vγ9Vδ2 T cells and aHSCs. We find that Vγ9Vδ2 T cells kill aHSCs and killing is enhanced when aHSCs are pretreated with BPH-1236, a lipophilic analog of the bone resorption drug zoledronate. Cytotoxicity is mediated by direct cell-to-cell contact as shown by Transwell experiments and atomic force microscopy, with BPH-1236 increasing the adhesion between aHSCs and Vγ9Vδ2 T cells. Mechanistically, BPH-1236 functions by inhibiting farnesyl diphosphate synthase, leading to accumulation of the phosphoantigen isopentenyl diphosphate and recognition by Vγ9Vδ2 T cells. The cytolytic process is largely dependent on the perforin/granzyme B pathway. In a Rag2−/−γc−/− immune-deficient mouse model, we find that Vγ9Vδ2 T cells home-in to the liver, and when accompanied by BPH-1236, kill not only orthotopic aHSCs but also orthotopic HCC tumors. Collectively, our results provide the first proof-of-concept of a novel immunotherapeutic strategy for the treatment of fibrosis–cirrhosis–HCC diseases using adoptively transferred Vγ9Vδ2 T cells, combined with a lipophilic bisphosphonate.

Inhibition of the mevalonate pathway augments the activity of pitavastatin against ovarian cancer cells

Only 40% of patients with advanced ovarian cancer survive more than 5 years. We have previously shown that pitavastatin induces regression of ovarian cancer xenografts in mice. To evaluate whether the response of ovarian cancer cells to pitavastatin is potentiated by farnesyl diphosphate synthase inhibitors or geranylgeraniol transferase I inhibitors, we evaluated combinations of pitavastatin with zoledronic acid, risedronate and GGTI-2133 in a panel of ovarian cancer cells. Pitavastatin (IC50 = 0.6–14 μM), zoledronic acid (IC50 = 21–57 μM), risedronate (IC50 > 100 μM) or GGTI-2133 (IC50 > 25 μM) inhibited the growth of ovarian cancer cell cultures. Combinations of pitavastatin with zoledronic acid displayed additive or synergistic effects in cell growth assays in 10 of 11 cell lines evaluated as well as in trypan blue exclusion, cellular ATP or caspase 3/7, 8 and 9 assays. Pitavastatin reduced levels of GGT-IIβ and the membrane localization of several small GTPases and this was potentiated by zoledronic acid. siRNA to GGT-Iβ and GGT-IIβ used in combination, but not when used individually, significantly increased the sensitivity of cells to pitavastatin. These data suggest that zoledronic acid, a drug already in clinical use, may be usefully combined with pitavastatin in the treatment of ovarian cancer.

Nano-technology based carriers for nitrogen-containing bisphosphonates delivery as sensitisers of γδ T cells for anticancer immunotherapy.

Nitrogen containing bisphosphonates (N-BPs) including zoledronate (ZOL) and alendronate (ALD) inhibit farnesyl diphosphate synthase, and have been shown to have a cytotoxic affect against cancer cells as a monotherapy and to also sensitise tumour cells to destruction by γδ T cells. γδ T cells are a subset of human T lymphocytes and have a diverse range of roles in the immune system including the recognition and destruction of cancer cells. This property of γδ T cells can be harnessed for use in cancer immunotherapy through in vivo expansion or the adoptive transfer of ex vivo activated γδ T cells. The use of N-BPs with γδ T cells has been shown to have a synergistic effect in in vitro, animal and clinical studies. N-BPs have limited in vivo activity due to rapid clearance from the circulation. By encapsulating N-BPs in liposomes (L) it is possible to increase the levels of N-BPs at non-osseous tumour sites. L-ZOL and L-ALD have been shown to have different toxicological profiles than free ZOL or ALD. Both L-ALD and L-ZOL led to increased spleen weight, leucocytosis, neutrophilia and lymphocytopenia in mice after intravenous injection. L-ALD was shown to be better tolerated than L-ZOL in murine studies. Biodistribution studies have been performed in order to better understand the interaction of N-BPs and γδ T cells in vivo. Additionally, in vivo therapy studies have shown that mice treated with both L-ALD and γδ T cells had a significant reduction in tumour growth compared to mice treated with L-ALD or γδ T cells alone. The use of ligand-targeted liposomes may further increase the efficacy of this combinatory immunotherapy. Liposomes targeting the αvβ6 integrin receptor using the peptide A20FMDV2 had a greater ability than untargeted liposomes in sensitising cancer cells to destruction by γδ T cells in αvβ6 positive cancer cell lines.

Geranylgeranyl diphosphate synthase inhibition induces apoptosis that is dependent upon GGPP depletion, ERK phosphorylation and caspase activation

Bisphosphonates are diphosphate analogs that inhibit the intermediate enzymes of the mevalonate pathway. Here, we compared the effects of a farnesyl diphosphate synthase inhibitor, zoledronate, and a geranylgeranyl diphosphate synthase (GGDPS) inhibitor, digeranyl bisphosphonate (DGBP), on lymphocytic leukemia cell proliferation and apoptosis. Both zoledronate and DGBP inhibited proliferation with DGBP doing so more potently. DGBP was markedly less toxic than zoledronate toward the viability of healthy human peripheral blood mononuclear cells. Addition of GGPP, but not farnesyl diphosphate (FPP), prevented the anti-proliferative effects of DGBP. Both GGPP and FPP partially rescued the effects of zoledronate. Co-treatment with DGBP and zoledronate was antagonistic. To further assess the effects of the bisphosphonates, we analyzed annexin V and propidium iodide staining via flow cytometry and found that DGBP induced apoptosis more potently than zoledronate. Western blots show that DGBP treatment altered expression and membrane affinity of some but not all geranylgeranylated small GTPases, activated caspases and increased ERK phosphorylation. Importantly, the anti-proliferative effects of DGBP were blocked by treatment with a caspase inhibitor and by treatment with a MEK inhibitor. Together, our findings indicate that DGBP is a more potent and selective compound than zoledronate in inducing apoptosis mediated through pathways that include caspases and MEK/ERK. These findings support the further development of GGDPS inhibitors as anticancer therapeutics.