Lentiviral-mediated silencing of farnesyl pyrophosphate synthase through RNA interference in mice.
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ABSTRACT:
SUBMITTER: Yang J
PROVIDER: S-EPMC4320928 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:To conform to the multiple regulations of triterpene biosynthesis, the gene encoding farnesyl pyrophosphate synthase (FPS) was transformed into Panax notoginseng (P. notoginseng) cells in which RNA interference (RNAi) of the cycloartenol synthase (CAS) gene had been accomplished. Transgenic cell lines showed both higher expression levels of FPS and lower expression levels of CAS compared to the wild-type (WT) cells. In the triterpene and phytosterol analysis, transgenic cell lines provided a higher accumulation of total triterpene saponins, and a lower amount of phytosterols in comparison with the WT cells. Compared with the cells in which RNAi of the CAS gene was achieved, the cells with simultaneously over-expressed FPS and silenced CAS showed higher triterpene contents. These results demonstrate that over-expression of FPS can break the rate-limiting reaction catalyzed by FPS in the triterpene saponins biosynthetic pathway; and inhibition of CAS expression can decrease the synthesis metabolic flux of the phytosterol branch. Thus, more precursors flow in the direction of triterpene synthesis, and ultimately promote the accumulation of P. notoginseng saponins. Meanwhile, silencing and over-expressing key enzyme genes simultaneously is more effective than just manipulating one gene in the regulation of saponin biosynthesis.
| S-EPMC6153935 | biostudies-literature
Project description:IntroductionFarnesyl pyrophosphate synthase (FPPS, also known as farnesyl diphosphate synthase (FDPS)) is one of the key enzymes involved in the mevalonate pathway and as such is widely expressed. FPPS modulators, specifically FPPS inhibitors, are useful in treating a number of diseases, including bone-related disorders characterized by excessive bone resorption, for example, osteoporosis, cancer metathesis to bone and infectious diseases caused by certain parasites.Areas coveredThis review covers structures and applications of novel FPPS modulators described in the patent literature from 2006 to 2010. Patents disclosing new formulations and uses of existing FPPS inhibitors are also reviewed. Thirty-three patents retrieved from the USPTO, EP and WIPO databases are examined with the goal of defining current trends in drug discovery related to FPPS inhibition, and its therapeutic effects.Expert opinionBisphosphonates (BPs) continue to dominate in this area, although other types of modulators are making their appearance. Remarkable for their high bone mineral affinity, BPs are structural mimics of the dimethylallyl pyrophosphate substrate of FPPS, and constitute the major type of FPPS inhibitor currently used in the clinic for treatment of bone-related diseases. Lipophilic BPs and new classes of non-BP FPPS inhibitors (salicylic acid and quinoline derivatives) have been introduced as possible alternatives for treatment of soft tissue diseases, such as some cancers. Novel formulations, fluorescent diagnostic probes and new therapeutic applications of existing FPPS inhibitors are also areas of significant patent activity, demonstrating growing recognition of the versatility and underdeveloped potential of these drugs.
| S-EPMC3510766 | biostudies-literature
Project description:Bisphosphonates (BPs) are the most widely used and effective treatment for osteoporosis and Paget's disease. Non-nitrogen containing BPs (non-N-BPs), namely etidronate, clodronate, tiludronate, as well as nitrogen containing BPs (N-BPs), namely pamidronate, alendronate, ibandronate, risedronate, zoledronate and minodronate have been launched on the market to date. N-BPs act by inhibiting the enzyme farnesyl pyrophosphate synthase (FPPS), and several crystal structures of complexes between FPPS and N-BPs have been revealed. Understanding the physical basis of the binding between protein and small molecules is an important goal in both medicinal chemistry and structural biology. In this review, we analyze in detail the energetic basis of molecular recognition between FPPS and N-BPs. First, we summarize the interactions between ligands and proteins observed in N-BPs-FPPS complexes in the Protein Data Bank (PDB). Second, we present an interaction energy analysis on the basis of full quantum mechanical calculation of FPPS and N-BP complexes using the fragment molecular orbital (FMO) method. The FMO result revealed that not only hydrogen bond and electrostatic interaction but also CH-O and π-π interaction with FPPS are important for N-BP's potency. Third, we describe a binding site analysis of FPPS on the basis of the inhomogeneous solvation theory which, by clustering the results from an explicit solvent molecular dynamics simulation (MD), is capable of describing the entropic and enthalpic contributions to the free energies of individual hydration sites. Finally, we also discuss the structure-activity relationship (SAR) of the series of minodronate derivatives.
| S-EPMC3343387 | biostudies-literature
Project description:Recently, several systems designed to trigger RNA interference by using small hairpin RNA driven by polymerase III promoters have been described. Here, we report a lentiviral-mediated small interfering RNA delivery system that can be induced by CRE recombinase. The system consists of a lentiviral vector carrying a mouse U6 promoter that is separated from a small hairpin RNA by a random DNA stuffer sequence flanked by modified loxP sites. The silencing cassette is not expressed until activated by addition of CRE recombinase delivered by a lentiviral vector. We have used this system to show specific down-regulation of GFP and two endogenous genes (the tumor suppressor p53 and the NF-kappaB transcription factor subunit p65) in vitro. Furthermore, down-regulation of both p53 and p65 resulted in the expected effect on downstream genes and cellular phenotype. We foresee multiple applications of this system both in vitro and in vivo to down-regulate specific targets in a tissue-specific and localized manner.
| S-EPMC409921 | biostudies-literature
Project description:Farnesylpyrophosphate synthase (FPS) catalyzes the biosynthesis of farnesyl pyrophosphate (FPP), which is an important precursor of sesquiterpenoids such as artemisinin and wilfordine. In the present study, we report the molecular cloning and characterization of two full-length cDNAs encoding FPSs from Tripterygium wilfordii (TwFPSs). TwFPSs maintained their capability to synthesise FPP in vitro when purified as recombinant proteins from E. coli. Consistent with the endogenous role of FPS in FPP biosynthesis, TwFPSs were highly expressed in T. wilfordii roots, and were up-regulated upon methyl jasmonate (MeJA) treatment. The global gene expression profiles suggested that the TwFPSs might play an important regulatory role interpenoid biosynthesis in T. wilfordii, laying the groundwork for the future study of the synthetic biology of natural terpene products.
| S-EPMC4418688 | biostudies-literature
Project description:Farnesyl pyrophosphate synthase (FPPS) is an enzyme of the mevalonate pathway and a well-established therapeutic target. Recent research has focused around a newly identified druggable pocket near the enzyme's active site. Pharmacological exploitation of this pocket is deemed promising; however, its natural biological function, if any, is yet unknown. Here we report that the product of FPPS, farnesyl pyrophosphate (FPP), can bind to this pocket and lock the enzyme in an inactive state. The Kd for this binding is 5-6 μM, within a catalytically relevant range. These results indicate that FPPS activity is sensitive to the product concentration. Kinetic analysis shows that the enzyme is inhibited through FPP accumulation. Having a specific physiological effector, FPPS is a bona fide allosteric enzyme. This allostery offers an exquisite mechanism for controlling prenyl pyrophosphate levels in vivo and thus contributes an additional layer of regulation to the mevalonate pathway.
| S-EPMC5253651 | biostudies-literature
Project description:Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways. Here we describe a novel role for this enzyme in fibroblast growth factor (FGF)-mediated signalling. We demonstrate the binding of FPPS to FGF receptors (FGFRs) using the yeast two-hybrid assay, pull-down assays and co-immunoprecipitation. The interaction between FPPS and FGFR is regulated by the cellular metabolic state and by treatment with FGF-2. Overexpression of FPPS inhibits FGF-2-induced cell proliferation, accompanied by a failure of the FGF-2-mediated induction of cyclins D1 and E. Overexpression of FPPS in fibroblasts also promotes increased farnesylation of Ras, and temporally extends FGF-2-stimulated activation of the Ras/ERK (extracellular-signal-regulated kinase) cascade. These data suggest that, in addition to its role in isoprenoid biosynthesis, FPPS may function as a modulator of the cellular response to FGF treatment.
| S-EPMC1222793 | biostudies-other
Project description:Farnesyl pyrophosphate synthase (FPS) belongs to the short-chain prenyltransferase family, and it performs a conserved and essential role in the terpenoid biosynthesis pathway. However, its classification, evolutionary history, and the forces driving the evolution of FPS genes in plants remain poorly understood.Phylogeny and positive selection analysis was used to identify the evolutionary forces that led to the functional divergence of FPS in plants, and recombinant detection was undertaken using the Genetic Algorithm for Recombination Detection (GARD) method. The dataset included 68 FPS variation pattern sequences (2 gymnosperms, 10 monocotyledons, 54 dicotyledons, and 2 outgroups). This study revealed that the FPS gene was under positive selection in plants. No recombinant within the FPS gene was found. Therefore, it was inferred that the positive selection of FPS had not been influenced by a recombinant episode. The positively selected sites were mainly located in the catalytic center and functional areas, which indicated that the 98S and 234D were important positively selected sites for plant FPS in the terpenoid biosynthesis pathway. They were located in the FPS conserved domain of the catalytic site. We inferred that the diversification of FPS genes was associated with functional divergence and could be driven by positive selection.It was clear that protein sequence evolution via positive selection was able to drive adaptive diversification in plant FPS proteins. This study provides information on the classification and positive selection of plant FPS genes, and the results could be useful for further research on the regulation of triterpenoid biosynthesis.
| S-EPMC5292144 | biostudies-literature
Project description:Mevalonate kinase is a key regulator of the mevalonate pathway, subject to feedback inhibition by the downstream metabolite farnesyl pyrophosphate. In this study, we validated the hypothesis that monophosphonate compounds mimicking farnesyl pyrophosphate can inhibit mevalonate kinase. Exploring compounds originally synthesized as allosteric inhibitors of farnesyl pyrophosphate synthase, we discovered mevalonate kinase inhibitors with nanomolar activity. Kinetic characterization of the two most potent inhibitors demonstrated Ki values of 3.1 and 22 nm. Structural comparison suggested features of these inhibitors likely responsible for their potency. Our findings introduce the first class of nanomolar inhibitors of human mevalonate kinase, opening avenues for future research. These compounds might prove useful as molecular tools to study mevalonate pathway regulation and evaluate mevalonate kinase as a potential therapeutic target.
| S-EPMC11301271 | biostudies-literature
Project description:Human farnesyl pyrophosphate synthase (hFPPS) catalyzes the production of the 15-carbon isoprenoid farnesyl pyrophosphate. The enzyme is a key regulator of the mevalonate pathway and a well-established drug target. Notably, it was elucidated as the molecular target of nitrogen-containing bisphosphonates, a class of drugs that have been widely successful against bone resorption disorders. More recently, research has focused on the anticancer effects of these inhibitors. In order to achieve increased non-skeletal tissue exposure, we created phenylaminopyridine bisphosphonates (PNP-BPs) that have bulky hydrophobic side chains through a structure-based approach. Some of these compounds have proven to be more potent than the current clinical drugs in a number of antiproliferation assays using multiple myeloma cell lines. In the present work, we characterized the binding of our most potent PNP-BPs to the target enzyme, hFPPS. Co-crystal structures demonstrate that the molecular interactions designed to elicit tighter binding are indeed established. We carried out thermodynamic studies as well; the newly introduced protein-ligand interactions are clearly reflected in the enthalpy of binding measured, which is more favorable for the new PNP-BPs than for the lead compound. These studies also indicate that the affinity of the PNP-BPs to hFPPS is comparable to that of the current drug risedronate. Risedronate forms additional polar interactions via its hydroxyl functional group and thus exhibits more favorable binding enthalpy; however, the entropy of binding is more favorable for the PNP-BPs, owing to the greater desolvation effects resulting from their large hydrophobic side chains. These results therefore confirm the overall validity of our drug design strategy. With a distinctly different molecular scaffold, the PNP-BPs described in this report represent an interesting new group of future drug candidates. Further investigation should follow to characterize the tissue distribution profile and assess the potential clinical benefits of these compounds.
| S-EPMC5643135 | biostudies-literature