Rational design of Lepidoptera-specific insecticidal inhibitors targeting farnesyl diphosphate synthase, a key enzyme of the juvenile hormone biosynthetic pathway.

Reducing the use of broad-spectrum insecticides is one of the many challenges currently faced by insect pest management practitioners. For this reason, efforts are being made to develop environmentally benign pest-control products through bio-rational approaches that aim at disrupting physiological processes unique to specific groups of pests. Perturbation of hormonal regulation of insect development and reproduction is one such strategy. It has long been hypothesized that some enzymes in the juvenile hormone biosynthetic pathway of moths, butterflies and caterpillars (order Lepidoptera) display unique structural features that could be targeted for the development of Lepidoptera-specific insecticides, a promising avenue given the numerous agricultural and forest pests belonging to this order. Farnesyl diphosphate synthase, FPPS, is one such enzyme, with recent work suggesting that it has structural characteristics that may enable its selective inhibition. This review synthesizes current knowledge on FPPS and summarizes recent advances in its use as a target for insecticide development.

Combination therapy in a xenograft model of glioblastoma: enhancement of the antitumor activity of temozolomide by an MDM2 antagonist.

OBJECTIVE Improvement in treatment outcome for patients with glioblastoma multiforme (GBM) requires a multifaceted approach due to dysregulation of numerous signaling pathways. The murine double minute 2 (MDM2) protein may fulfill this requirement because it is involved in the regulation of growth, survival, and invasion. The objective of this study was to investigate the impact of modulating MDM2 function in combination with front-line temozolomide (TMZ) therapy in GBM. METHODS The combination of TMZ with the MDM2 protein-protein interaction inhibitor nutlin3a was evaluated for effects on cell growth, p53 pathway activation, expression of DNA repair proteins, and invasive properties. In vivo efficacy was assessed in xenograft models of human GBM. RESULTS In combination, TMZ/nutlin3a was additive to synergistic in decreasing growth of wild-type p53 GBM cells. Pharmacodynamic studies demonstrated that inhibition of cell growth following exposure to TMZ/nutlin3a correlated with: 1) activation of the p53 pathway, 2) downregulation of DNA repair proteins, 3) persistence of DNA damage, and 4) decreased invasion. Pharmacokinetic studies indicated that nutlin3a was detected in human intracranial tumor xenografts. To assess therapeutic potential, efficacy studies were conducted in a xenograft model of intracranial GBM by using GBM cells derived from a recurrent wild-type p53 GBM that is highly TMZ resistant (GBM10). Three 5-day cycles of TMZ/nutlin3a resulted in a significant increase in the survival of mice with GBM10 intracranial tumors compared with single-agent therapy. CONCLUSIONS Modulation of MDM2/p53-associated signaling pathways is a novel approach for decreasing TMZ resistance in GBM. To the authors' knowledge, this is the first study in a humanized intracranial patient-derived xenograft model to demonstrate the efficacy of combining front-line TMZ therapy and an inhibitor of MDM2 protein-protein interactions.

Disruption of insect isoprenoid biosynthesis with pyridinium bisphosphonates.

Farnesyl diphosphate synthase (FPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C5), with the allylic diphosphate primer dimethylallyl diphosphate (DMAPP; C5) to generate the C15 prenyl chain (FPP) used for protein prenylation as well as sterol and terpene biosynthesis. Here, we designed and prepared a series of pyridinium bisphosphonate (PyrBP) compounds, with the aim of selectively inhibiting FPPS of the lepidopteran insect order. FPPSs of Drosophila melanogaster and the spruce budworm, Choristoneura fumiferana, were inhibited by several PyrBPs, and as hypothesized, larger bisphosphonates were more selective for the lepidopteran protein and completely inactive towards dipteran and vertebrate FPPSs. Cell growth of a D. melanogaster cell line was adversely affected by exposure to PyrPBs that were strongly inhibitory to insect FPPS, although their effect was less pronounced than that observed upon exposure to the electron transport disrupter, chlorfenapyr. To assess the impact of PyrBPs on lepidopteran insect growth and development, we performed feeding and topical studies, using the tobacco hornworm, Manduca sexta, as our insect model. The free acid form of a PyrBP and a known bisphosphonate inhibitor of vertebrate FPPS, alendronate, had little to no effect on larval M. sexta; however, the topical application of more lipophilic ester PyrBPs caused decreased growth, incomplete larval molting, cuticle darkening at the site of application, and for those insects that survived, the formation of larval-pupal hybrids. To gain a better understanding of the structural differences that produce selective lepidopteran FPPS inhibition, homology models of C. fumiferana and D. melanogaster FPPS (CfFPPS2, and DmFPPS) were prepared. Docking of substrates and PyrBPs demonstrates that differences at the -3 and -4 positions relative to the first aspartate rich motif (FARM) are important factors in the ability of the lepidopteran enzyme to produce homologous isoprenoid structure and to be selectively inhibited by larger PyrBPs.

Cloning, expression and characterization of an insect geranylgeranyl diphosphate synthase from Choristoneura fumiferana.

Geranylgeranyl diphosphate synthase (GGPPS) catalyzes the condensation of the non-allylic diphosphate, isopentenyl diphosphate (IPP; C5), with allylic diphosphates to generate the C20 prenyl chain (GGPP) used for protein prenylation and diterpenoid biosynthesis. Here, we cloned the cDNA of a GGPPS from the spruce budworm, Choristoneura fumiferana, and characterized the corresponding recombinant protein (rCfGGPPS). As shown for other type-III GGPPSs, rCfGGPPS preferred farnesyl diphosphate (FPP; C15) over other allylic substrates for coupling with IPP. Unexpectedly, rCfGGPPS displayed inhibition by its FPP substrate at low IPP concentration, suggesting the existence of a mechanism that may regulate intracellular FPP pools. rCfGGPPS was also inhibited by its product, GGPP, in a competitive manner with respect to FPP, as reported for human and bovine brain GGPPSs. A homology model of CfGGPPS was prepared and compared to human and yeast GGPPSs. Consistent with its enzymological properties, CfGGPPS displayed a larger active site cavity that can accommodate the binding of FPP and GGPP in the region normally occupied by IPP and the allylic isoprenoid tail, and the binding of GGPP in an alternate orientation seen for GGPP binding to the human protein. To begin exploring the role of CfGGPPS in protein prenylation, its transcripts were quantified by qPCR in whole insects, along with those of other genes involved in this pathway. CfGGPPS was expressed throughout insect development and the abundance of its transcripts covaried with that of other prenylation-related genes. Our qPCR results suggest that geranylgeranylation is the predominant form of prenylation in whole C. fumiferana.

Cloning, expression and characterization of lepidopteran isopentenyl diphosphate isomerase.

Isopentenyl diphosphate isomerase (IPPI) of the spruce budworm, Choristoneura fumiferana, and of the tobacco hornworm, Manduca sexta, was cloned and its catalytic properties assessed. In the presence of Mg(2+) or Mn(2+), the recombinant protein from C. fumiferana (CfIPPI) efficiently isomerized IPP to dimethylallyl diphosphate (DMAPP). While C. fumiferana IPPI transcript levels were evenly distributed in a wide variety of tissues, they were highly abundant in the corpora allata. Because IPPI plays an alternate role in lepidopteran juvenile hormone (JH) biosynthesis by catalyzing the isomerization of the homologous substrate, homoisopentenyl diphosphate (HIPP), the ability of CfIPPI to convert HIPP to homodimethylallyl diphosphate (HDMAPP) was also studied. As expected, HIPP isomerization was efficient and the formation of HDMAPP occurred, but the regiospecificity of the reaction was lower than previously found in M. sexta corpora allata homogenates and with purified Bombyx mori IPPI. Differences in inhibitory potency for several alkylated ammonium diphosphates and higher homologs of DMAPP were noted between CfIPPI and a vertebrate IPPI, suggesting that the lepidopteran enzyme has a larger active site cavity. To determine the structural factors responsible for homologous substrate coupling, site directed mutagenesis of several residues identified through sequence alignment and homology modeling analysis was performed. The results suggest that unlike other IPPIs, W216 (C. fumiferana numbering) works in concert with a tyrosine residue (Y105) to allow binding of larger substrates and to stabilize the high-energy intermediate formed during substrate isomerization.

Farnesylcysteine lyase is involved in negative regulation of abscisic acid signaling in Arabidopsis.

The Arabidopsis FCLY gene encodes a specific farnesylcysteine (FC) lyase, which is responsible for the oxidative metabolism of FC to farnesal and cysteine. In addition, fcly mutants with quantitative decreases in FC lyase activity exhibit an enhanced response to ABA. However, the enzymological properties of the FCLY-encoded enzyme and its precise role in ABA signaling remain unclear. Here, we show that recombinant Arabidopsis FC lyase expressed in insect cells exhibits high selectivity for FC as a substrate and requires FAD and molecular oxygen for activity. Arabidopsis FC lyase is also shown to undergo post-translational N-glycosylation. FC, which is a competitive inhibitor of isoprenylcysteine methyltransferase (ICMT), accumulates in fcly mutants. Moreover, the enhanced response of fcly mutants to ABA is reversed by ICMT overexpression. These observations support the hypothesis that the ABA hypersensitive phenotype of fcly plants is the result of FC accumulation and inhibition of ICMT.

Effect of trans unsaturation on molecular organization in a phospholipid membrane.

We employed solid state (2)H NMR, complemented by computer simulations, to compare molecular organization in model membranes composed of 1-elaidoyl-2-stearoylphosphatidylcholine (t18:1-18:0PC), 1-oleoyl-2-stearoylphosphatidylcholine (c18:1-18:0PC), and 1,2-distearoylphosphatidylcholine (18:0-18:0PC). These phospholipids have elaidic acid (EA) containing a trans double bond, oleic acid (OA) containing a cis double bond, and saturated stearic acid (SA), respectively, at the sn-1 position and were synthesized with perdeuterated SA at the sn-2 position. The temperature of the chain melting transition is depressed less for t18:1-18:0PC (31.5 degrees C) than c18:1-18:0PC (7 degrees C) relative to 18:0-18:0PC (53 degrees C), reflecting the smaller deviation from the linear conformation produced by a trans as opposed to cis double bond. Acyl chain order in t18:1-18:0PC (S(CD) = 0.135) in the liquid crystalline state is much closer to that of c18:1-18:0PC (S(CD) = 0.128) than that of the substantially more ordered 18:0-18:0PC (S(CD) > 0.156), which is attributed to the reduced energy barrier to rotation about the C-C single bonds next to either a trans or cis carbon double bond. A conformation that somewhat resembles a saturated chain and an intrinsic disorder approaching that of a cis unsaturated chain characterize EA and, we speculate, may play a role in the adverse impact dietary trans fatty acids (TFA) have on biological function.

Characterization of a novel aphid prenyltransferase displaying dual geranyl/farnesyl diphosphate synthase activity.

We report on the cDNA cloning and characterization of a novel short-chain isoprenyl diphosphate synthase from the aphid Myzus persicae. Of the three IPPS cDNAs we cloned, two yielded prenyltransferase activity following expression in Escherichia coli; these cDNAs encode identical proteins except for the presence, in one of them, of an N-terminal mitochondrial targeting peptide. Although the aphid enzyme was predicted to be a farnesyl diphosphate synthase by BLASTP analysis, rMpIPPS, when isopentenyl diphosphate and dimethylallyl diphosphate are supplied as substrates, typically generated geranyl diphosphate (C10) as its main product, along with significant quantities of farnesyl diphosphate (C15). Analysis of an MpIPPS homology model pointed to substitutions that could confer GPP/FPP synthase activity to the aphid enzyme.

Cloning, expression and characterization of a dipteran farnesyl diphosphate synthase.

Farnesyl diphosphate synthase (FPPS) of the dipteran Drosophila melanogaster has been cloned and its catalytic properties have been assessed. Analysis of the D. melanogaster genome and of ESTs indicates that FPPS is a single copy gene that produces two transcripts, which differ only by 5' extension. The cDNA of shorter and longer D. melanogaster FPPSs (DmFPPS-1a and DmFPPS-1b, respectively) were each subcloned into pET28a and expressed as an N-His6 fusion protein in BL21 E. coli cells. The DmFPPSs similarly catalyzed the coupling of the allylic substrates, GPP and DMAPP, with IPP, producing FPP as product. The longer protein was further characterized. The enzyme required divalent metal for activity, and was activated by 0.1% Triton X-100. Higher detergent concentration and the addition of glycerol, conditions that activate certain insect FPPSs, inhibited prenyl coupling by DmFPPS-1b. Although DmFPPS-1b does not efficiently couple homologous GPP compounds, homodimethylallyl diphosphate (HDMAPP), which is precursor to all homologous JH structures, was a reactive substrate.

Purification, properties and heteromeric association of type-1 and type-2 lepidopteran farnesyl diphosphate synthases.

Two forms of farnesyl diphosphate synthase (FPPS) from the spruce budworm, Choristoneura fumiferana, and one from the armyworm Pseudaletia unipuncta, have been cloned and their catalytic properties assessed. The type-2 FPPS of C. fumiferana (CfFPPS2) was efficient in the prenyl coupling of DMAPP or GPP with [(14)C]IPP, producing FPP as its final product; however, type-1 FPPSs (CfFPPS1, PuFPPS1, as well as Agrotis ipsilon FPPS1) were essentially inactive. A variety of purification methods was employed to purify the type-1 enzymes. Under mild chromatographic conditions, the isolated type-1 enzyme showed modest activity, but was apparently contaminated with endogenous prenyltransferase derived from the Escherichia coli host cells. Similarly, unpurified extracts of PuFPPS1 expressed in an E. coli FPPS-null mutant, had low FPPS activity. When equimolar amounts of homogenous CfFPPS1 and CfFPP2 were combined, a sharp synergistic enhancement of activity was observed, and the coupling of several homologous substrates, which are precursors to ethyl-branched JHs, was enhanced. Association between CfFPPS1 and CfFPPS2 was confirmed by both protein interaction chromatography and competitive ELISA. These data suggest that type-1 and type-2 FPPSs can form a heteromer, which may play a role in sesquiterpene biosynthesis, such as JH homologue formation, in moths.

Arabidopsis thaliana plants possess a specific farnesylcysteine lyase that is involved in detoxification and recycling of farnesylcysteine.

In plants, prenylated proteins are involved in actin organization, calcium-mediated signal transduction, and many other biological processes. Arabidopsis thaliana mutants lacking functional protein prenyltransferase genes have also revealed roles for prenylated proteins in phytohormone signaling and meristem development. However, to date, the turnover of prenylated plant proteins and the fate of the prenylcysteine (PC) residue have not been described. We have detected an enzyme activity in Arabidopsis plants that metabolizes farnesylcysteine (FC) to farnesal, which is subsequently reduced to farnesol. Unlike its mammalian ortholog, Arabidopsis FC lyase exhibits specificity for FC over geranylgeranylcysteine (GGC), and recognizes N-acetyl-FC (AFC). FC lyase is encoded by a gene on chromosome 5 of the Arabidopsis genome (FCLY, At5g63910) and is ubiquitously expressed in Arabidopsis tissues and organs. Furthermore, T-DNA insertions into the FCLY gene cause significant decreases in FC lyase activity and an enhanced response to abscisic acid (ABA) in seed germination assays. The effects of FCLY mutations on ABA sensitivity are even greater in the presence of exogenous FC. These data suggest that plants possess a specific FC detoxification and recycling pathway.

Prenyltransferase of larval and adult M. sexta corpora allata.

Prenyltransferase activity derived from the corpora allata (CA) of the lepidopteran insect, Manduca sexta, has been characterized. The coupling of allylic substrates DMAPP and GPP with the non-allylic substrate IPP was evaluated using CA homogenates of both the larval and adult stages of development. The effect of additives and inhibitors, assay conditions, and metal preference were examined. The cellular location of prenyltransferase activity was also investigated. We found subtle differences between larval and adult preparations, including metal and detergent preference, and while larval prenyltransferase activity was strictly cytosolic, prenyltransferase derived from adult CA was found in both the cytosolic and pellet fractions. Differences in kinetics as a function of development were also noted. When GPP was utilized as allylic substrate, adult prenyltransferase displayed cooperative behavior; while with DMAPP, biphasic kinetics were observed. In fifth instar larvae, prenyltransferase activity was highest on days 1-2 and reaction end products changed as a result of insect age. Taken together, these results suggest that larval and adult prenyltransferase of M. sexta have distinct enzymological properties and that the adult CA possess more than one prenyltransferase.

Juvenile hormone biosynthesis in M. sexta: substrate specificity of insect prenyltransferase utilizing homologous diphosphate analogs.

Analogs of dimethylallyl diphosphate (DMAPP) and geranyl diphosphate (GPP) were prepared and tested as potential substrates of prenyltransferase of the tobacco hornworm, Manduca sexta, and of a sesquiterpene synthase derived from pig liver. Enzyme derived from corpora allata homogenates of both the larval and adult stage of M. sexta coupled each of the DMAPP analogs to produce homologous geranyl and farnesyl diphosphate products in the order (Z)-3-ethyl>(Z)-3-n-propyl>(Z)-3-methyl (DMAPP)>(Z)-3-i-propyl(Z)-3-n-butyl. In competition studies, the ethyl and n-propyl analogs either enhanced or had no effect on DMAPP coupling, whereas the larger analogs were inhibitors. (Z)-7-ethyl and (2Z,6Z)-3,7-diethyl analogs of GPP were as good, if not better substrates of larval prenyltransferase, while the C-3 ethyl analog of GPP, which is precursor to an isomeric form of juvenile hormone (JH) that is not typically found in insects, was poorly coupled by the enzyme. While similarities were seen for whole-cell extracts derived from adult and larval M. sexta, adult prenyltransferase derived from cytosolic and 16,000xg pellet fractions displayed distinct competitive coupling of GPP and its homologs, suggesting differences in substrate specificity as a result of enzyme localization. In contrast to M. sexta, the pig liver enzyme poorly coupled each of the homologous DMAPP derivatives, and the homologous derivatives of GPP were less efficiently coupled than GPP. These results indicate that prenyltransferase in M. sexta possesses high steric latitude at the (Z)-C-3 and C-7 alkyl positions of DMAPP and GPP, respectively, in contrast to other animal prenyltransferases but in keeping with the enzyme's presumptive role in homologous JH metabolism.

Characterization and tissue-specific expression of two lepidopteran farnesyl diphosphate synthase homologs: implications for the biosynthesis of ethyl-substituted juvenile hormones.

The sesquiterpenoid juvenile hormone (JH) regulates insect development and reproduction. Most insects produce only one chemical form of JH, but the Lepidoptera produce four derivatives featuring ethyl branches. The biogenesis of these JHs requires the synthesis of ethyl-substituted farnesyl diphosphate (FPP) by FPP synthase (FPPS). To determine if there exist more than one lepidopteran FPPS, and whether one FPPS homolog is better adapted for binding the bulkier ethyl-branched substrates/products, we cloned three lepidopteran FPPS cDNAs, two from Choristoneura fumiferana and one from Pseudaletia unipuncta. Amino acid sequence comparisons among these and other eukaryotic FPPSs led to the recognition of two lepidopteran FPPS types. Type-I FPPSs display unique active site substitutions, including several in and near the first aspartate-rich motif, whereas type-II proteins have a more "conventional" catalytic cavity. In a yeast assay, a Drosophila FPPS clone provided full complementation of an FPPS mutation, but lepidopteran FPPS clones of either type yielded only partial complementation, suggesting unusual catalytic features and/or requirements of these enzymes. Although a structural analysis of lepidopteran FPPS active sites suggested that type-I enzymes are better suited than type-II for generating ethyl-substituted products, a quantitative real-time PCR assessment of their relative abundance in insect tissues indicated that type-I expression is ubiquitous whereas that of type-II is essentially confined to the JH-producing glands, where its transcripts are approximately 20 times more abundant than those of type-I. These results suggest that type-II FPPS plays a leading role in lepidopteran JH biosynthesis in spite of its apparently more conventional catalytic cavity.

Partial purification of a farnesyl diphosphate synthase from whole-body Manduca sexta.

Farnesyl diphosphate synthase (FPP synthase) is a ubiquitous enzyme that is required for the biosynthesis of sesquiterpenes, dolichols ubiquinones, and prenylated proteins in insects. We report on the partial purification and characterization of an FPP synthase, obtained from whole-body preparations of the lepidopteran insect, Manduca sexta. The larval enzyme was separated from isopentenyl diphosphate (IPP) isomerase, phosphatase, and GGPP synthase by preparative isoelectric focusing, and was further purified by DEAE Sepharose, hydroxyapatite, and size exclusion chromatography. Whole-body M. sexta FPP synthase has a native molecular weight of 60.5+/-3.5 kDa and consists of two subunits of 28.5+/-0.5 kDa. As seen with other prenyltransferases, the enzyme has an absolute requirement for divalent cation and both Mn(2+) and Mg(2+) stimulated activity, although the former was inhibitory at higher concentrations. Insect FPP synthase catalyzes the condensation of IPP (K(m)=2.9+/-1.2 microM) with both dimethylallyl diphosphate and geranyl diphosphate (K(m)=0.8+/-0.4 microM). The enzyme requires the presence of detergent, glycerol, and non-specific protein-protein interactions for stability and maximum catalytic activity.

Biomimetic Cyclization of Enamide-Containing Polyenes as a New Route to Azapolycycles.

Enamide 4 was studied for its effectiveness as a polyene precursor in biomimetic cyclizations. While most conventional Lewis acids were poor cyclization promoters, FeCl(3).6H(2)O initiated the conversion of 4 into tricycles 6 and 7 in excellent yield. The two isomeric products result from the cyclization of intermediate aldehyde 5 by either a chair or boat B-ring transition state. These results suggest that enamides may be incorporated into polyene precursors for the construction of larger azapolycycles such as azasteroids.