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David F. Wiemer - One of the best experts on this subject based on the ideXlab platform.
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quantitative determination of a potent Geranylgeranyl Diphosphate Synthase inhibitor using lc ms ms derivatization and application
Journal of Pharmaceutical and Biomedical Analysis, 2018Co-Authors: Yashpal S Chhonker, David F. Wiemer, Sarah A. Holstein, Staci L Haney, Robert A Matthiesen, Daryl J MurryAbstract: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.
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Stereoselective Synthesis of Homoneryl and Homogeranyl Triazole Bisphosphonates
2016Co-Authors: Robert A. Matthiesen, Sarah A. Holstein, Veronica S. Wills, Joseph I. Metzger, David F. WiemerAbstract:Isoprenoid-substituted bisphosphonates are known to serve as inhibitors of the enzyme Geranylgeranyl Diphosphate Synthase, and their activity can be highly sensitive to olefin stereochemistry. A mixture of homogeranyl and homoneryl triazole bisphosphonates has previously demonstrated potent activity, and thus stereocontrolled syntheses of the individual isomers have been developed
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Potent Triazole Bisphosphonate Inhibitor of Geranylgeranyl Diphosphate Synthase
2015Co-Authors: Veronica S. Wills, Cheryl Allen, Sarah A. Holstein, David F. WiemerAbstract:Studies of triazole bisphosphonates have resulted in identification of a potent inhibitor of Geranylgeranyl Diphosphate Synthase (IC50 = 45 nM) with very good selectivity for this enzyme over farnesyl Diphosphate Synthase (IC50 = 28 μM). This compound also potently disrupts Geranylgeranylation and induces cytotoxicity in human myeloma cells at submicromolar levels, suggesting that it may serve as a lead compound for treatment of malignancies characterized by excessive protein secretion
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a novel class of Geranylgeranyl Diphosphate Synthase inhibitors structure activity relationships of homoisoprenoid triazoles in myeloma cells
Blood, 2014Co-Authors: Veronica S. Wills, David F. Wiemer, Xiang Zhou, E Born, Cheryl Allen, Sarah A. HolsteinAbstract:The secretion of monoclonal protein and the dependence on the surrounding bone marrow microenvironment are key features of malignant plasma cells. We have previously demonstrated that targeting the Rab family of small GTPases via disruption of the post-translational modification Geranylgeranylation not only impairs intracellular monoclonal protein trafficking, thus leading to endoplasmic reticulum stress and apoptosis, but also disrupts the interaction of the malignant plasma cells with key components of the bone marrow microenvironment. While developing agents that directly target the enzyme that Geranylgeranylates the Rabs, we discovered several compounds that specifically and potently inhibit the enzyme Geranylgeranyl Diphosphate Synthase (GGDPS). This enzyme is responsible for synthesis of the isoprenoid substrate which is used in Geranylgeranylation reactions, thus inhibition of this enzyme is an alternative approach by which to target Rabs. Families of compounds were therefore designed which incorporated a hydrophobic chain, a triazole ring, and a bisphosphonic acid head group. The central triazole provides a potential zinc-binding element, and allows for divergent assembly of potential inhibitors through click chemistry. Through this strategy, prenyl, homoprenyl, geranyl, homogeranyl, farnesyl, homofarnesyl, Geranylgeranyl, and homoGeranylgeranyl triazole bisphosphonates have been prepared. All of these agents were subjected to in vitro enzyme assays for GGDPS as well as for the related enzyme farnesyl Diphosphate Synthase (FDPS), and IC50’s were determined. Cellular activity in human myeloma cell lines (RPMI-8226, U266, MM.1S) was assessed via immunoblot analysis of representative prenylated proteins. These studies revealed that in all cases, the homologated-version of the allylic triazole is more potent than the corresponding isoprenoid against GGDPS, ranging from 1.1-fold improvement in the homoGeranylgeranyl:Geranylgeranyl pairing to almost 300-fold in the homogeranyl:geranyl pairing. Chain length was found to be an important factor which determines the inhibitory activity within the homoisoprenoid series: the order of increasing potency against GGDPS was homoGeranylgeranyl Disclosures No relevant conflicts of interest to declare.
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Synthesis of isoprenoid bisphosphonate ethers through C–P bond formations: Potential inhibitors of Geranylgeranyl Diphosphate Synthase
Beilstein journal of organic chemistry, 2014Co-Authors: Xiang Zhou, Raymond J. Hohl, Jacqueline E. Reilly, Kathleen A. Loerch, David F. WiemerAbstract:A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme Geranylgeranyl Diphosphate Synthase (GGDPS). Five of the new compounds show IC50 values of less than 1 μM against GGDPS with little to no activity against the related enzyme farnesyl Diphosphate Synthase (FDPS). The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 μM concentration.
Emilia A. Kimura - One of the best experts on this subject based on the ideXlab platform.
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Plasmodium falciparum parasites overexpressing farnesyl Diphosphate Synthase/Geranylgeranyl Diphosphate Synthase are more resistant to risedronate
Memorias do Instituto Oswaldo Cruz, 2018Co-Authors: Heloisa B. Gabriel, Mauro F Azevedo, Emilia A. Kimura, Alejandro M. KatzinAbstract:Farnesyl Diphosphate Synthase/Geranylgeranyl Diphosphate Synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains. Risedronate, a bisphosphonate containing nitrogen (N-BP), is a potent inhibitor of blood stage Plasmodium. Here, we show that P. falciparum parasites overexpressing FPPS/GGPPS are more resistant to risedronate, suggesting that this enzyme is an important target, and bisphosphonate analogues can be used as potential antimalarial drugs.
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plasmodium falciparum parasites overexpressing farnesyl Diphosphate Synthase Geranylgeranyl Diphosphate Synthase are more resistant to risedronate
Memorias Do Instituto Oswaldo Cruz, 2018Co-Authors: Heloisa B. Gabriel, Mauro F Azevedo, Emilia A. Kimura, Alejandro M. KatzinAbstract:Farnesyl Diphosphate Synthase/Geranylgeranyl Diphosphate Synthase (FPPS/GGPPS) is a key enzyme in the synthesis of isoprenic chains. Risedronate, a bisphosphonate containing nitrogen (N-BP), is a potent inhibitor of blood stage Plasmodium. Here, we show that P. falciparum parasites overexpressing FPPS/GGPPS are more resistant to risedronate, suggesting that this enzyme is an important target, and bisphosphonate analogues can be used as potential antimalarial drugs.
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Cloning and characterization of bifunctional enzyme farnesyl Diphosphate/Geranylgeranyl Diphosphate Synthase from Plasmodium falciparum
Malaria journal, 2013Co-Authors: Fabiana Morandi Jordão, Heloisa B. Gabriel, Mauro F Azevedo, João M. P. Alves, Claudia Blanes Angeli, Thaís D. Bifano, Ardala Breda, Luiz Augusto Basso, Gerhard Wunderlich, Emilia A. KimuraAbstract:Background: Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol Diphosphate pathway and the products are further metabolized by farnesyl Diphosphate Synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods: The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4- 14 C] isopentenyl Diphosphate and three different allylic substrates: dimethylallyl Diphosphate, geranyl Diphosphate or farnesyl Diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results: The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl Diphosphate, as well as Geranylgeranyl Diphosphate, being therefore a bifunctional FPPS/Geranylgeranyl Diphosphate Synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl Diphosphate, geranyl Diphosphate and farnesyl Diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions: The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.
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cloning and characterization of bifunctional enzyme farnesyl Diphosphate Geranylgeranyl Diphosphate Synthase from plasmodium falciparum
Malaria Journal, 2013Co-Authors: Fabiana Morandi Jordão, Heloisa B. Gabriel, Mauro F Azevedo, João M. P. Alves, Claudia Blanes Angeli, Thaís D. Bifano, Ardala Breda, Luiz Augusto Basso, Gerhard Wunderlich, Emilia A. KimuraAbstract:Background: Isoprenoids are the most diverse and abundant group of natural products. In Plasmodium falciparum, isoprenoid synthesis proceeds through the methyl erythritol Diphosphate pathway and the products are further metabolized by farnesyl Diphosphate Synthase (FPPS), turning this enzyme into a key branch point of the isoprenoid synthesis. Changes in FPPS activity could alter the flux of isoprenoid compounds downstream of FPPS and, hence, play a central role in the regulation of a number of essential functions in Plasmodium parasites. Methods: The isolation and cloning of gene PF3D7_18400 was done by amplification from cDNA from mixed stage parasites of P. falciparum. After sequencing, the fragment was subcloned in pGEX2T for recombinant protein expression. To verify if the PF3D7_1128400 gene encodes a functional rPfFPPS protein, its catalytic activity was assessed using the substrate [4- 14 C] isopentenyl Diphosphate and three different allylic substrates: dimethylallyl Diphosphate, geranyl Diphosphate or farnesyl Diphosphate. The reaction products were identified by thin layer chromatography and reverse phase high-performance liquid chromatography. To confirm the product spectrum formed of rPfFPPS, isoprenic compounds were also identified by mass spectrometry. Apparent kinetic constants KM and Vmax for each substrate were determined by Michaelis–Menten; also, inhibition assays were performed using risedronate. Results: The expressed protein of P. falciparum FPPS (rPfFPPS) catalyzes the synthesis of farnesyl Diphosphate, as well as Geranylgeranyl Diphosphate, being therefore a bifunctional FPPS/Geranylgeranyl Diphosphate Synthase (GGPPS) enzyme. The apparent KM values for the substrates dimethylallyl Diphosphate, geranyl Diphosphate and farnesyl Diphosphate were, respectively, 68 ± 5 μM, 7.8 ± 1.3 μM and 2.06 ± 0.4 μM. The protein is expressed constitutively in all intra-erythrocytic stages of P. falciparum, demonstrated by using transgenic parasites with a haemagglutinin-tagged version of FPPS. Also, the present data demonstrate that the recombinant protein is inhibited by risedronate. Conclusions: The rPfFPPS is a bifunctional FPPS/GGPPS enzyme and the structure of products FOH and GGOH were confirmed mass spectrometry. Plasmodial FPPS represents a potential target for the rational design of chemotherapeutic agents to treat malaria.
Ran Wang - One of the best experts on this subject based on the ideXlab platform.
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heteromeric Geranylgeranyl Diphosphate Synthase contributes to carotenoid biosynthesis in ripening fruits of red pepper capsicum annuum var conoides
Journal of Agricultural and Food Chemistry, 2018Co-Authors: Qiang Wang, Xingqi Huang, Tian-jun Cao, Zhong Zhuang, Ran WangAbstract:Pepper ( Capsicum annuum) fruits are a rich source of carotenoids. Geranylgeranyl Diphosphate (GGPP) is the precursor for carotenoid biosynthesis and is produced by GGPP Synthase (GGPPS), which belongs to the prenyl transferase (PTS) family. In this study, we identified from the pepper genome a total of eight PTS homologues. Our subcellular localization, enzymatic activity, and expression level analyses proved that among these homologues Capana04g000412 is the only functional GGPPS (CaGGPPS1) for carotenoid biosynthesis in pepper fruits. We demonstrated that CaGGPPS1 interacts with a catalytically inactive small subunit homologue protein CaSSUII, and such an interaction promotes CaGGPPS1 enzymatic activity. We also revealed a protein-protein interaction between CaSSUII and a putative phytoene Synthase and the repression of carotenoid accumulation by silencing CaSSUII in pepper fruits. Taken together, our results suggest an essential contribution of the CaGGPPS1/CaSSUII interaction to carotenoid biosynthesis in ripening pepper fruits.
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Heteromeric Geranylgeranyl Diphosphate Synthase Contributes to Carotenoid Biosynthesis in Ripening Fruits of Red Pepper (Capsicum annuum var. conoides)
2018Co-Authors: Qiang Wang, Xingqi Huang, Tian-jun Cao, Zhong Zhuang, Ran WangAbstract:Pepper (Capsicum annuum) fruits are a rich source of carotenoids. Geranylgeranyl Diphosphate (GGPP) is the precursor for carotenoid biosynthesis and is produced by GGPP Synthase (GGPPS), which belongs to the prenyl transferase (PTS) family. In this study, we identified from the pepper genome a total of eight PTS homologues. Our subcellular localization, enzymatic activity, and expression level analyses proved that among these homologues Capana04g000412 is the only functional GGPPS (CaGGPPS1) for carotenoid biosynthesis in pepper fruits. We demonstrated that CaGGPPS1 interacts with a catalytically inactive small subunit homologue protein CaSSUII, and such an interaction promotes CaGGPPS1 enzymatic activity. We also revealed a protein–protein interaction between CaSSUII and a putative phytoene Synthase and the repression of carotenoid accumulation by silencing CaSSUII in pepper fruits. Taken together, our results suggest an essential contribution of the CaGGPPS1/CaSSUII interaction to carotenoid biosynthesis in ripening pepper fruits
Silvia N. J. Moreno - One of the best experts on this subject based on the ideXlab platform.
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the farnesyl Diphosphate Geranylgeranyl Diphosphate Synthase of toxoplasma gondii is a bifunctional enzyme and a molecular target of bisphosphonates
Journal of Biological Chemistry, 2007Co-Authors: Yan Ling, Eric Oldfield, Kildare Miranda, Silvia N. J. MorenoAbstract:Farnesyl-Diphosphate Synthase (FPPS) catalyzes the synthesis of farnesyl Diphosphate, an important precursor of sterols, dolichols, ubiquinones, and prenylated proteins. We report the cloning and characterization of two Toxoplasma gondii farnesyl-Diphosphate Synthase (TgFPPS) homologs. A single genetic locus produces two transcripts, TgFPPS and TgFPPSi, by alternative splicing. Both isoforms were heterologously expressed in Escherichia coli, but only TgFPPS was active. The protein products predicted from the nucleotide sequences have 646 and 605 amino acids and apparent molecular masses of 69.5 and 64.5 kDa, respectively. Several conserved sequence motifs found in other prenyl-Diphosphate Synthases are present in both TgFPPSs. TgFPPS was also expressed in the baculovirus system and was biochemically characterized. In contrast to the FPPS of other eukaryotic organisms, TgFPPS is bifunctional, catalyzing the formation of both farnesyl Diphosphate and Geranylgeranyl Diphosphate. TgFPPS localizes to the mitochondria, as determined by the co-localisation of the affinity-purified antibodies against the protein with MitoTracker, and in accord with the presence of an N-terminal mitochondria-targeting signal in the protein. This enzyme is an attractive target for drug development, because the order of inhibition of the enzyme by a number of bisphosphonates is the same as that for inhibition of parasite growth. In summary, we report the first bifunctional farnesyl-Diphosphate/Geranylgeranyl-Diphosphate Synthase identified in eukaryotes, which, together with previous results, establishes this enzyme as a valid target for the chemotherapy of toxoplasmosis.
Xiang Zhou - One of the best experts on this subject based on the ideXlab platform.
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a novel class of Geranylgeranyl Diphosphate Synthase inhibitors structure activity relationships of homoisoprenoid triazoles in myeloma cells
Blood, 2014Co-Authors: Veronica S. Wills, David F. Wiemer, Xiang Zhou, E Born, Cheryl Allen, Sarah A. HolsteinAbstract:The secretion of monoclonal protein and the dependence on the surrounding bone marrow microenvironment are key features of malignant plasma cells. We have previously demonstrated that targeting the Rab family of small GTPases via disruption of the post-translational modification Geranylgeranylation not only impairs intracellular monoclonal protein trafficking, thus leading to endoplasmic reticulum stress and apoptosis, but also disrupts the interaction of the malignant plasma cells with key components of the bone marrow microenvironment. While developing agents that directly target the enzyme that Geranylgeranylates the Rabs, we discovered several compounds that specifically and potently inhibit the enzyme Geranylgeranyl Diphosphate Synthase (GGDPS). This enzyme is responsible for synthesis of the isoprenoid substrate which is used in Geranylgeranylation reactions, thus inhibition of this enzyme is an alternative approach by which to target Rabs. Families of compounds were therefore designed which incorporated a hydrophobic chain, a triazole ring, and a bisphosphonic acid head group. The central triazole provides a potential zinc-binding element, and allows for divergent assembly of potential inhibitors through click chemistry. Through this strategy, prenyl, homoprenyl, geranyl, homogeranyl, farnesyl, homofarnesyl, Geranylgeranyl, and homoGeranylgeranyl triazole bisphosphonates have been prepared. All of these agents were subjected to in vitro enzyme assays for GGDPS as well as for the related enzyme farnesyl Diphosphate Synthase (FDPS), and IC50’s were determined. Cellular activity in human myeloma cell lines (RPMI-8226, U266, MM.1S) was assessed via immunoblot analysis of representative prenylated proteins. These studies revealed that in all cases, the homologated-version of the allylic triazole is more potent than the corresponding isoprenoid against GGDPS, ranging from 1.1-fold improvement in the homoGeranylgeranyl:Geranylgeranyl pairing to almost 300-fold in the homogeranyl:geranyl pairing. Chain length was found to be an important factor which determines the inhibitory activity within the homoisoprenoid series: the order of increasing potency against GGDPS was homoGeranylgeranyl Disclosures No relevant conflicts of interest to declare.
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synthesis of isoprenoid bisphosphonate ethers through c p bond formations potential inhibitors of Geranylgeranyl Diphosphate Synthase
Beilstein Journal of Organic Chemistry, 2014Co-Authors: Xiang Zhou, Raymond J. Hohl, Jacqueline E. Reilly, Kathleen A. Loerch, David F. WiemerAbstract:A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme Geranylgeranyl Diphosphate Synthase (GGDPS). Five of the new compounds show IC50 values of less than 1 μM against GGDPS with little to no activity against the related enzyme farnesyl Diphosphate Synthase (FDPS). The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 μM concentration.
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Synthesis of isoprenoid bisphosphonate ethers through C–P bond formations: Potential inhibitors of Geranylgeranyl Diphosphate Synthase
Beilstein journal of organic chemistry, 2014Co-Authors: Xiang Zhou, Raymond J. Hohl, Jacqueline E. Reilly, Kathleen A. Loerch, David F. WiemerAbstract:A set of bisphosphonate ethers has been prepared through sequential phosphonylation and alkylation of monophosphonate ethers. After formation of the corresponding phosphonic acid salts, these compounds were tested for their ability to inhibit the enzyme Geranylgeranyl Diphosphate Synthase (GGDPS). Five of the new compounds show IC50 values of less than 1 μM against GGDPS with little to no activity against the related enzyme farnesyl Diphosphate Synthase (FDPS). The most active compound displayed an IC50 value of 82 nM when assayed with GGDPS, and no activity against FDPS even at a 10 μM concentration.
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geranyl and neryl triazole bisphosphonates as inhibitors of Geranylgeranyl Diphosphate Synthase
Bioorganic & Medicinal Chemistry, 2014Co-Authors: Xiang Zhou, Huaxiang Tong, David F. Wiemer, Veronica S. Wills, Sarah D Ferree, E Born, Sarah A. HolsteinAbstract:When inhibitors of enzymes that utilize isoprenoid pyrophosphates are based on the natural substrates, a significant challenge can be to achieve selective inhibition of a specific enzyme. One element in the design process is the stereochemistry of the isoprenoid olefins. We recently reported preparation of a series of isoprenoid triazoles as potential inhibitors of Geranylgeranyl transferase II but these compounds were obtained as a mixture of olefin isomers. We now have accomplished the stereoselective synthesis of these triazoles through the use of epoxy azides for the cycloaddition reaction followed by regeneration of the desired olefin. Both geranyl and neryl derivatives have been prepared as single olefin isomers through parallel reaction sequences. The products were assayed against multiple enzymes as well as in cell culture studies and surprisingly a Z-olefin isomer was found to be a potent and selective inhibitor of Geranylgeranyl Diphosphate Synthase.