Structural Insight into Geranylgeranyl Diphosphate Synthase (GGDPS) for Cancer Therapy.

Geranylgeranyl diphosphate synthase (GGDPS), the source of the isoprenoid donor in protein geranylgeranylation reactions, has become an attractive target for anticancer therapy due to the reliance of cancers on geranylgeranylated proteins. Current GGDPS inhibitor development focuses on optimizing the drug-target enzyme interactions of nitrogen-containing bisphosphonate-based drugs. To advance GGDPS inhibitor development, understanding the enzyme structure, active site, and ligand/product interactions is essential. Here we provide a comprehensive structure-focused review of GGDPS. We reviewed available yeast and human GGDPS structures and then used AlphaFold modeling to complete unsolved structural aspects of these models. We delineate the elements of higher-order structure formation, product-substrate binding, the electrostatic surface, and small-molecule inhibitor binding. With the rise of structure-based drug design, the information provided here will serve as a valuable tool for rationally optimizing inhibitor selectivity and effectiveness.

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.

Perillyl alcohol attenuates chronic restraint stress aggravated dextran sulfate sodium-induced ulcerative colitis by modulating TLR4/NF-κB and JAK2/STAT3 signaling pathways.

BACKGROUND: Ulcerative colitis (UC) is the most prevalent chronic inflammatory immune bowel disease. The modernization of lifestyle accompanied by the stress to cope with the competition has resulted in a new range of complications where stress became a critical contributing factor for many diseases, including UC. Hence there is an urgent need to develop a dual role in curtailing both systemic and neuroinflammation. Perillyl alcohol (POH) is a natural essential oil found in lavender, peppermint, cherries etc and has been widely studied for its strong anti-inflammatory, antioxidant and anti-stress properties. HYPOTHESIS/PURPOSE: POH regulates the various inflammatory signaling cascades involved in chronic inflammation by inhibiting farnesyltransferase  enzyme. Several studies reported that POH could inhibit the phosphorylation of  NF-κB, STAT3 and promote the endogenous antioxidant enzymes like Nrf2 via farnesyltransferase enzyme inhibition.  Also, the effects of POH against UC is not known yet. Thus, this study aims to explore the anti-ulcerative properties of POH on stress aggravated ulcerative colitis in C57BL/6 mice. METHODS: Ulcerative colitis was induced by duel exposure of chronic restraint stress (day 1 to day 28) and 2.5% dextran sulphate sodium (day8 to day14) in mice. POH treatment 100 and 200 mg/kg was administred from day14 ti day28 following oral route of administration. Disease activity index, colonoscopy, western blot analysis and histological analysis, neurotransmitter analysis and Gene expression studies were perofomerd to asses the anti-colitis effects of POH. RESULTS: The treatment reversed the oxidative stress and inflammatory response by inhibiting TLR4/NF-kB pathway, and IL-6/JAK2/STAT3 pathway in both isolated mice colons and brains. The inhibition of these pathways resulted in a decrease in pro-inflammatory cytokines like IL-6, IL-1ß and TNF-α. The treatment improved the physiological and histological changes with decreased ulcerations as examined by colonic endoscopy and Haematoxylin and Eosin staining. The treatment also improved the behavior response as it increased mobility time which was reduced by chronic restrained stress. This was due to increased satiety neurotransmitters like dopamine and serotonin and decreased cortisol in mice brains. CONCLUSION: These results infer that POH has significant anti-colitis activity on chronic restraint stress aggravated DSS-induced UC in mice.

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.

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.

The spatio-temporal expression of some genes involved in the biosynthetic pathways of terpenes/phenylpropanoids in yarrow (Achillea millefolium).

Yarrow (Achillea millefolium) is a medicinal plant from the Asteracea which biosynthesize different secondary metabolites especially terpenes and phenylpropanoids. To improve our understanding of the regulatory mechanisms behind the biosynthesis of these compounds we analyzed the expression of some genes associated with the biosynthesis of terpenes and phenylpropanoids in different tissues and in response to trans-cinnamic acid (tCA) as an inhibitor of PAL activity. Isolation and expression analysis of DXR, GPPS, PAL and CHS genes together with linalool synthase (LIS) as monoterpene synthase was conducted in different developmental stages of leaves, flowers and in response to trans-cinnamic acid (tCA). Differential expression of these genes observed in different tissues. tCA up-regulated the biosynthetic genes of monterpenes and down-regulated the biosynthetic genes of phenylpropanoids. Gene expression analysis in intact leaves and leaves without glandular trichomes showed that DXR, LIS, PAL and CHS are highly expressed in glandular trichomes while GPPS expressed ubiquitously. Analysis of essential oils composition showed that sesquiterpenes and monoterpenes are main compounds; in which from 57 identified compounds the highest were germacreneD (% 11.5), guaiol (%10.38), spatulenol (%8.73) and caryophyllene oxide (%7.48).

Bioactivity of Farnesyltransferase Inhibitors Against Entamoeba histolytica and Schistosoma mansoni.

The protozoan parasite Entamoeba histolytica can induce amebic colitis and amebic liver abscess. First-line drugs for the treatment of amebiasis are nitroimidazoles, particularly metronidazole. Metronidazole has side effects and potential drug resistance is a concern. Schistosomiasis, a chronic and painful infection, is caused by various species of the Schistosoma flatworm. There is only one partially effective drug, praziquantel, a worrisome situation should drug resistance emerge. As many essential metabolic pathways and enzymes are shared between eukaryotic organisms, it is possible to conceive of small molecule interventions that target more than one organism or target, particularly when chemical matter is already available. Farnesyltransferase (FT), the last common enzyme for products derived from the mevalonate pathway, is vital for diverse functions, including cell differentiation and growth. Both E. histolytica and Schistosoma mansoni genomes encode FT genes. In this study, we phenotypically screened E. histolytica and S. mansoni in vitro with the established FT inhibitors, lonafarnib and tipifarnib, and with 125 tipifarnib analogs previously screened against both the whole organism and/or the FT of Trypanosoma brucei and Trypanosoma cruzi. For E. histolytica, we also explored whether synergy arises by combining lonafarnib and metronidazole or lonafarnib with statins that modulate protein prenylation. We demonstrate the anti-amebic and anti-schistosomal activities of lonafarnib and tipifarnib, and identify 17 tipifarnib analogs with more than 75% growth inhibition at 50 µM against E. histolytica. Apart from five analogs of tipifarnib exhibiting activity against both E. histolytica and S. mansoni, 10 additional analogs demonstrated anti-schistosomal activity (severe degenerative changes at 10 µM after 24 h). Analysis of the structure-activity relationship available for the T. brucei FT suggests that FT may not be the relevant target in E. histolytica and S. mansoni. For E. histolytica, combination of metronidazole and lonafarnib resulted in synergism for growth inhibition. Also, of a number of statins tested, simvastatin exhibited moderate anti-amebic activity which, when combined with lonafarnib, resulted in slight synergism. Even in the absence of a definitive molecular target, identification of potent anti-parasitic tipifarnib analogs encourages further exploration while the synergistic combination of metronidazole and lonafarnib offers a promising treatment strategy for amebiasis.

Inhibition of farnesyl pyrophosphate (FPP) and/or geranylgeranyl pyrophosphate (GGPP) biosynthesis and its implication in the treatment of cancers.

Dysregulation of isoprenoid biosynthesis is implicated in numerous biochemical disorders that play a role in the onset and/or progression of age-related diseases, such as hypercholesterolemia, osteoporosis, various cancers, and neurodegeneration. The mevalonate metabolic pathway is responsible for the biosynthesis of the two key isoprenoid metabolites, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Post-translational prenylation of various proteins, including the small GTP-binding proteins (GTPases), with either FPP or GGPP is vital for proper localization and activation of these proteins. Prenylated GTPases play a critical role in cell signaling, proliferation, cellular plasticity, oncogenesis, and cancer metastasis. Pre-clinical and clinical studies strongly suggest that inhibition of protein prenylation can be an effective treatment for non-skeletal cancers. In this review, we summarize the most recent drug discovery efforts focusing on blocking protein farnesylation and/or geranylgeranylation and the biochemical and structural data available in guiding the current on-going studies in drug discovery. Furthermore, we provide a summary on the biochemical association between disruption of protein prenylation, endoplasmic reticulum (ER) stress, unfolded protein response (UPR) signaling, and cancer.

Blue light decreases tanshinone IIA content in Salvia miltiorrhiza hairy roots via genes regulation.

The effect of light-emitting diodes (LEDs) on the production of secondary metabolites in medicinal plants and hairy roots is receiving much attention. The roots and rhizomes of the traditional Chinese medicinal plant Salvia miltiorrhiza Bunge are widely used for treating cardiovascular and cerebrovascular diseases. The main components are liposoluble tanshinones and hydrophilic phenolic acids. Moreover, hairy root culture of S. miltiorrhiza has been used in research of valuable plant-derived secondary metabolites. In this study, we examined the effect of LEDs with different combinations of wavelengths on the content of the main components in hairy roots of S. miltiorrhiza. Tanshinone IIA (TSIIA) content in hairy roots was significantly decreased with all light treatments containing blue light by >60% and was 9 times lower with LED treatment duration changed from 1 week to 3 weeks. HMGR, DXS2, DXR, GGPPS, CPS and CYP76AH1 genes involved in the tanshinone biosynthesis pathway were downregulated by blue light. Furthermore, light quality treatments have different effect on the accumulation of phenolic acids in hairy roots of S. miltiorrhiza. The light treatments 6R3B, 6B3IR, 7RGB and 2R6BUV for 3 weeks could increase rosmarinic acid (RA) content slightly but not salvianolic acid B (SAB) content. Different secondary metabolite contents could be regulated by different wavelength combinations of LEDs. Blue light could reduce TSIIA content in hairy roots of S. miltiorrhiza via gene regulation.

Diverse responses of tanshinone biosynthesis to biotic and abiotic elicitors in hairy root cultures of Salvia miltiorrhiza and Salvia castanea Diels f. tomentosa.

Salvia miltiorrhiza (S. miltiorrhiza) and Salvia castanea Diels f. tomentosa (S. castanea) are both used for treatment of cardiovascular diseases. They have the same bioactive compound tanshinones, but whose contents are hugely different. This study illustrated diverse responses of tanshinone biosynthesis to yeast extract (YE) and Ag+ in hairy roots of the two species. YE enhanced both the growth and tanshinone biosynthesis of two hairy roots, and contributed more to tanshinone accumulation in S. castanea than that in S. miltiorrhiza. Genes encoding 1-deoxy-d-xylulose 5-phosphate synthase (DXS2), geranylgeranyl diphosphatesynthase (GGPPS1), copalyl diphosphate synthase (CPS1), and two cytochromes P450 (CYP76AH1 and CYP76AH3) were also more responsive to YE in S. castanea than those in S. miltiorrhiza. Accumulations of dihydrotanshinone I and tanshinone I, and most biosynthetic genes in S. miltiorrhiza were more responsive to Ag+ than those in S. castanea. Accumulations of dihydrotanshinone I and cryptotanshinone were more responsive to YE, while tanshinone IIA accumulation was more responsive to Ag+ in S. miltiorrhiza. However, accumulations of other four tanshinones and related genes in S. castanea were more responsive to YE than Ag+. This study provides foundations for studying diverse specialized metabolism between the related species.

[Cloning and bioinformatics analysis of geranylgeranyl diphosphate synthase gene of Tripterygium wilfordii].

A full-length cDNA of GGPPS gene from Tripterygium wilfordii suspension cells was obtained by use of RACE strategy (GeneBank: KM978333), and then analyzed by bioinformatics approaches. TwGGPPS cDNA has 1857 nucleotides and an open reading frame (ORF) encoding a protein of 514 amino acid residues. The deduced protein has isoelectric point (pI) of 7.85, a calculated molecular weight about 57.13 kD, 5 conserved domains and 2 functional domains. PSORT Prediction showed it was located at plasma membrane. Phylogenetic analysis demonstrated that TwGGPPS1 was similar to GGPPS from other species of plants. For the first time the cloning of geranylgeranyl diphosphate synthase gene from T. wilfordii was reported, it lays the foundation for further research of diterpenoids biosynthetic pathway.

Transcriptional activation of a geranylgeranyl diphosphate synthase gene, GGPPS2, isolated from Scoparia dulcis by treatment with methyl jasmonate and yeast extract.

A cDNA clone, designated SdGGPPS2, was isolated from young seedlings of Scoparia dulcis. The putative amino acid sequence of the translate of the gene showed high homology with geranylgeranyl diphosphate synthase (GGPPS) from various plant sources, and the N-terminal residues exhibited the characteristics of chloroplast targeting sequence. An appreciable increase in the transcriptional level of SdGGPPS2 was observed by exposure of the leaf tissues of S. dulcis to methyl jasmonate, yeast extract or Ca(2+) ionophore A23187. In contrast, SdGGPPS1, a homologous GGPPS gene of the plant, showed no or only negligible change in the expression level upon treatment with these stimuli. The truncated protein heterologously expressed in Escherichia coli in which the putative targeting domain was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to liberate geranylgeranyl diphosphate. These results suggested that SdGGPPS2 plays physiological roles in methyl jasmonate and yeast extract-induced metabolism in the chloroplast of S. dulcis cells.

Three types of geranylgeranyl diphosphate synthases from the medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

Geranylgeranyl diphosphate synthase (GGPPS) is a key enzyme in the carotenoid biosynthetic pathway, catalyzing the synthesis of its C20 precursor. In the present study, three types of ggpps genes were cloned and analyzed from the Caterpillar Medicinal Fungus Cordyceps militaris, a valued carotenoid-producing species. The sequences were named as ggpps727, ggpps191, and ggpps595. The open reading frame codes for predicted polypeptides of 464, 550, and 431 aa. Three predicted GGPPSs had a high similarity to that from Beauveria bassiana ARSEF 2860 with identity of 73%, 71%, and 56%, respectively. Homology comparison of the deduced peptide sequences of the various GGPPSs revealed highly conserved domains. Both GGPPS727 and GGPPS191 from C. militaris contained all five domains highly conserved among prenyltransferases as well as two aspartate-rich DDXX(XX)D motifs in domains II and V, which have been proven essential for prenyltransferase activity. By constructing the phylogenetic tree of fungal GGPPSs, it was found that fungi-derived GGPPSs could be divided into three clusters, suggesting there were three types of GGPPSs in fungi. Each type may be responsible for a different metabolism. Three types of GGPPSs from C. militaris belonged to the different clusters separately. Expression analysis of three ggpps genes during the fruit body cultivation of C. militaris by real-time polymerase chain reaction (PCR) suggested the ggpps 191 gene may be involved in the synthesis of carotenoids and ggpps 727 may be responsible for primary metabolism. This is the first report of the GGPPS from C. militaris, a valued edible and medicinal fungus.

Moiety-linkage map reveals selective nonbisphosphonate inhibitors of human geranylgeranyl diphosphate synthase.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS). Current bisphosphonate drugs (e.g., Fosamax and Zometa) are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease, and tumor-induced osteolysis, but they are often less potent in blood and soft-tissue due to their phosphate moieties. The discovery of nonbisphosphonate inhibitors of FPPS and/or GGPPS for the treatment of bone diseases and cancers is, therefore, a current goal. Here, we propose a moiety-linkage-based method, combining a site-moiety map with chemical structure rules (CSRs), to discover nonbisphosphonate inhibitors from thousands of commercially available compounds and known crystal structures. Our moiety-linkage map reveals the binding mechanisms and inhibitory efficacies of 51 human GGPPS (hGGPPS) inhibitors. To the best of our knowledge, we are the first team to discover two novel selective nonbisphosphonate inhibitors, which bind to the inhibitory site of hGGPPS, using CSRs and site-moiety maps. These two compounds can be considered as a novel lead for the potent inhibitors of hGGPPS for the treatment of cancers and mevalonate-pathway diseases. Moreover, based on our moiety-linkage map, we identified two key residues of hGGPPS, K202, and K212, which play an important role for the inhibitory effect of zoledronate (IC50 = 3.4 µM and 2.4 µM, respectively). This result suggests that our method can discover specific hGGPPS inhibitors across multiple prenyltransferases. These results show that the compounds that highly fit our moiety-linkage map often inhibit hGGPPS activity and induce tumor cell apoptosis. We believe that our method is useful for discovering potential inhibitors and binding mechanisms for pharmaceutical targets.

Characterization and subcellular localization of geranylgeranyl diphosphate synthase from Catharanthus roseus.

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

Farnesyltransferase inhibitor FTI-277 reduces mortality of septic mice along with improved bacterial clearance.

Treatment with statins, inhibitors of HMG-CoA reductase, extends the survival of septic mice. However, the molecular mechanisms underlying the cholesterol-lowering, independent beneficial effects of statins in sepsis are poorly understood. The inhibition of protein isoprenylation, namely farnesylation and geranylgeranylation, has been proposed as a mediator of the pleiotropic protective effects of statins, although direct evidence is lacking. Major features of sepsis-induced immune suppression include T-cell dysfunction, which is characterized by apoptosis of splenic T cells, increased CD4(+)Foxp3(+) regulatory T cells (Tregs), and suppression of type 1 helper T-cell response [e.g., interferon-γ (IFN-γ) secretion] in mice. Here, we show that the induction of sepsis by cecal ligation and puncture (CLP) resulted in increases in farnesyltransferase activity and farnesylated proteins in the spleen relative to sham operation. Treatment with farnesyltransferase inhibitor N-[4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl]methionine methyl ester trifluoroacetate salt (FTI-277) (25 mg/kg b.wt. i.p.) at 2 h after CLP blocked the increase in farnesylated proteins and improved survival and bacterial clearance of septic mice. FTI-277 reverted to or mitigated sepsis-induced apoptosis in spleen and thymus, increased splenic CD4(+)Foxp3(+) Tregs, and suppressed IFN-γ secretion and proliferation of splenocytes in response to anti-CD3+CD28 antibodies in mice. Moreover, FTI-277 promoted macrophage phagocytotic activity in septic mice. These results indicate that elevation in protein farnesylation plays a role in derangements in immune function and mortality of septic mice. These findings suggest that prevention of immune dysfunction might contribute to FTI-277-induced improvement in survival of septic mice. These data highlight protein farnesyltransferase as a novel potential molecular target to reduce the mortality of patients with sepsis.

Thiazole- and imidazole-containing peptidomimetic inhibitors of protein farnesyltransferase.

Mimetics of the C-terminal CAAX tetrapeptide of Ras protein were designed replacing internal dipeptide AA with 4-amino-2-phenylbenzoic acid and cysteine (C) with 2-amino-4-thiazolyl-, 2-mercapto-4-thiazolyl-, 2-mercapto-4-imidazolyl- and 2-methylmercapto-4-thiazolyl-acetic or propionic acid. The compound in which C is replaced by 2-amino-4-thiazolylacetic acid inhibited FTase activity in the low nanomolar range and showed antiproliferative effect on rat aortic smooth muscle cells interfering with Ras farnesylation. On the basis of these results, 2-aminothiazole can be considered as an alternative to heterocycles, such as pyridine and imidazole, normally used in FTase inhibitors designed as non-thiol CAAX mimetics.

Driven to death: Inhibition of farnesylation increases Ras activity and promotes growth arrest and cell death [corrected].

To improve cancer outcomes, investigators are turning increasingly to small molecule medicines that disrupt vital signaling cascades, inhibit malignant growth, or induce apoptosis. One vital signaling molecule is Ras, and a key step in Ras activation is membrane anchoring of Ras through prenylation, the C-terminal addition of a lipid anchor. Small molecule inhibitors of farnesyltransferase (FTI), the enzyme most often responsible for prenylating Ras, showed clinical promise, but development of FTIs such as tipifarnib has been stalled by uncertainty about their mechanism of action, because Ras seemed unimpeded in tipifarnib-treated samples. Interpretation was further complicated by the numerous proteins that may be farnesylated, as well as availability of an alternate prenylation pathway, geranylgeranylation. Our initial observations of varied response by cancer cell lines to tipifarnib led us to evaluate the role of FTI in Ras signal alteration using various tumor models. We describe our novel counterintuitive finding that endogenous Ras activity increases in cancer cell lines with low endogenous Ras activity when farnesyltransferase is inhibited by either tipifarnib or short hairpin RNA. In response to tipifarnib, variable growth arrest and/or cell death correlated with levels of activated extracellular signal­regulated kinase (ERK) and p38 mitogenactivated protein kinase (MAPK). Sensitivity to tipifarnib treatment was shown by growth inhibition and by an increase in subdiploid cell numbers; cells with such sensitivity had increased activation of ERK and p38 MAPK. Because Ras must be prenylated to be active, our findings suggest that geranylgeranylated N-Ras or K-Ras B interacts differently with downstream effector proteins in sensitive cancer cells responding to tipifarnib, switching the balance from cell proliferation to growth inhibition [corrected].

Cloning and expression of 1-deoxy-d-xylulose 5-phosphate synthase cDNA from Croton stellatopilosus and expression of 2C-methyl-d-erythritol 4-phosphate synthase and geranylgeranyl diphosphate synthase, key enzymes of plaunotol biosynthesis.

1-Deoxy-d-xylulose 5-phosphate synthase (DXS, EC: 4.1.3.37), the first enzyme in the 2C-methyl-d-erythritol 4-phosphate (MEP) pathway, is known to be responsible for the rate-limiting step of isoprenoid biosynthesis in Escherichia coli and Arabidopsis thaliana. In this study, the dxs gene from Croton stellatopilosus, designated csdxs, was cloned from leaf tissue using the rapid amplification of cDNA ends (RACE) technique. Leaves of C. stellatopilosus contain plaunotol, an acyclic diterpene alcohol. The csdxs cDNA containing the open reading frame of 2163 base pairs appeared to encode a polypeptide of 720 amino acids. Analysis of the deduced amino acid sequence revealed that the NH(2)-terminus of CSDXS carried a chloroplast transit peptide, a thiamine diphosphate binding site, and a transketolase motif, which are the important characteristics of DXS enzymes in higher plants. Multiple alignments of CSDXS with other plant DXSs have indicated that CSDXS has identity ranging between 68% and 89%. Expression levels of csdxs and genes encoding key enzymes in the plaunotol biosynthetic pathway, namely 2C-methyl-d-erythritol 4-phosphate synthase (meps) and geranylgeranyl diphosphate synthase (ggpps), were analysed by measuring transcript levels in leaves of different developmental stages. The results showed that dxs, meps, and ggpps are all active in young leaves prior to full expansion when plaunotol is synthesised from the DXP precursor in chloroplasts. The dense presence of chloroplasts and oil globules in the palisade cells of these leaves support the view that these genes are involved in plaunotol biosynthesis in chloroplast-containing tissues.

A bifunctional geranyl and geranylgeranyl diphosphate synthase is involved in terpene oleoresin formation in Picea abies.

The conifer Picea abies (Norway spruce) defends itself against herbivores and pathogens with a terpenoid-based oleoresin composed chiefly of monoterpenes (C(10)) and diterpenes (C(20)). An important group of enzymes in oleoresin biosynthesis are the short-chain isoprenyl diphosphate synthases that produce geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)), and geranylgeranyl diphosphate (C(20)) as precursors of different terpenoid classes. We isolated a gene from P. abies via a homology-based polymerase chain reaction approach that encodes a short-chain isoprenyl diphosphate synthase making an unusual mixture of two products, geranyl diphosphate (C(10)) and geranylgeranyl diphosphate (C(20)). This bifunctionality was confirmed by expression in both prokaryotic (Escherichia coli) and eukaryotic (P. abies embryogenic tissue) hosts. Thus, this isoprenyl diphosphate synthase, designated PaIDS1, could contribute to the biosynthesis of both major terpene types in P. abies oleoresin. In saplings, PaIDS1 transcript was restricted to wood and bark, and transcript level increased dramatically after methyl jasmonate treatment, which induces the formation of new (traumatic) resin ducts. Polyclonal antibodies localized the PaIDS1 protein to the epithelial cells surrounding the traumatic resin ducts. PaIDS1 has a close phylogenetic relationship to single-product conifer geranyl diphosphate and geranylgeranyl diphosphate synthases. Its catalytic properties and reaction mechanism resemble those of conifer geranylgeranyl diphosphate synthases, except that significant quantities of the intermediate geranyl diphosphate are released. Using site-directed mutagenesis and chimeras of PaIDS1 with single-product geranyl diphosphate and geranylgeranyl diphosphate synthases, specific amino acid residues were identified that alter the relative composition of geranyl to geranylgeranyl diphosphate.