Synthesis of a potential photoactivatable anandamide analog

A potential photoreactive analog of anandamide was synthesized via selective hydrogenation of a skipped tetrayne intermediate. This compound might be a useful tool to search for new cannabinoid receptors.     

A 5′-amino analog of adenosine diphosphate

The synthesis and properties of the 5′-amino analog of adenosine diphosphate are described. The 5′-amino-5′-deoxyadenosine 5′-N-diphosphate (_NADP) is prepared from the previously described aminonucleoside triphosphate by the hexokinase catalyzed transfer of the terminal phosphoryl to glucose. The _NADP is stable in neutral or basic media, and is similar to natural ADP in chromatographic, electrophoretic, and spectrographic properties. Snake venom phosphodiesterase degrades _NADP to the monophosphate _NAMP, and acid degrades both _NADP and _NAMP to 5′-amino-5′-deoxyadenosine.     

Temperature-dependent modulation of farnesyl diphosphate/geranylgeranyl diphosphate synthase from hyperthermophilic archaea

Enzyme characteristics of trans-prenyl diphosphate synthase (Tk-IdsA) from Thermococcus kodakaraensis, which catalyzes the consecutive trans-condensation of isopentenyl diphosphate (C(5)) units with allylic diphosphate, were examined. Product analysis revealed that Tk-IdsA is a bifunctional enzyme, farnesyl diphosphate (FPP, C(15))/geranylgeranyl diphosphate (GGPP, C(20)) synthase, and mainly yields both C(15) and C(20). The FPP/GGPP product ratio increases with the rise of the reaction temperature. The kinetic parameters obtained at 70 and 90 degrees C demonstrated that the rise of the temperature elevates the k(0) value for the C(10) allylic substrate to more than those for the C(5) and C(15) allylic substrates. These data suggest that Tk-IdsA contributes to adjust the membrane composition to the cell growth temperature by modulating its substrate and product specificities. Mutation study indicated that the aromatic side chain of Tyr-81 acts as a steric hindrance to terminate the chain elongation and defines the final product length.     

Purification of geranylgeranyl diphosphate synthase from Phycomyces blakesleanus

Geranylgeranyl diphosphate synthase has been purified to homogeneity from the carotene-overproducing strain M1 of Phycomyces blakesleanus. Usually two activity peaks with molecular weights of 60,000 and 30,000 eluted on gel exclusion chromatography, suggesting that the enzyme consists of two subunits, with a tendency to dissociate. With homogeneous protein, a single-staining band with molecular weight of 30,000 appeared on sodium dodecyl sulfate gel electrophoresis, confirming a subunit molecular weight of 30,000. Only isopentenyl diphosphate and farnesyl diphosphate were accepted by this enzyme for geranylgeranyl diphosphate formation. The smaller allylic compounds, dimethylallyl and geranyl diphosphate, were utilized at less than 1/20th the rate of farnesyl diphosphate. Michaelis constants of 9 microM for isopentenyl diphosphate and 60 microM for farnesyl diphosphate were found. The isoelectric point is 4.8.     

Stable analogues of geranylgeranyl diphosphate possessing improved geranylgeranyl versus farnesyl protein transferase inhibitory selectivity

Phosphonoacetamido(oxy) groups have proven to be good mimics of the diphosphate portion in geranylgeranyl protein transferase I (GGTase I) inhibitors. The introduction of small alkyl groups (Me, Et) into the diphosphate mimic moiety caused a further decrease in collateral farnesyl protein transferase (FTase) inhibitory activity, thereby improving GGTase I over FTase selectivity.     

Amides as bioisosteres of triazole-based geranylgeranyl diphosphate synthase inhibitors

Geranylgeranyl diphosphate synthase (GGDPS) inhibitors are of potential therapeutic interest as a consequence of their activity against the bone marrow cancer multiple myeloma. A series of bisphosphonates linked to an isoprenoid tail through an amide linkage has been prepared and tested for the ability to inhibit GGDPS in enzyme and cell-based assays. The amides were designed as analogues to triazole-based GGDPS inhibitors. Several of the new compounds show GGDPS inhibitory activity in both enzyme and cell assays, with potency dependent on chain length and olefin stereochemistry.     

Interaction of yeast Rab geranylgeranyl transferase with its protein and lipid substrates

Small GTPases from the Rab/Ypt family regulate events of vesicular traffic in eukaryotic cells. For their activity, Rab proteins require a posttranslational modification that is conferred by Rab geranylgeranyltransferase (RabGGTase), which attaches geranylgeranyl moieties onto two cysteines of their C terminus. RabGGTase is present in both lower and higher eukaryotes in the form of heterodimers composed of alpha and beta subunits. However, the alpha subunits of RabGGTases from lower eukaryotes, including Saccharomyces cerevisiae (yRabGGTase), are half the size of the corresponding subunit of the mammalian enzyme. This difference is due to the presence of additional immunoglobulin (Ig)-like and leucine rich (LRR) domains in the mammalian transferase. To understand the possible evolutionary implications and functional consequences of structural differences between RabGGTases of higher and lower eukaryotes, we have investigated the interactions of yeast RabGGTase with its lipid and protein substrate. We have demonstrated that geranylgeranyl pyrophosphate binds to the enzyme with an affinity of ca. 40 nM, while binding of farnesyl pyrophosphate is much weaker, with a K(d) value of ca. 750 nM. This finding suggests that despite the structural difference, yRabGGTase selects its lipid substrate in a fashion similar to mammalian RabGGTase. However, unlike the mammalian enzyme, yRabGGTase binds prenylated and unprenylated Ypt1p:Mrs6p complexes with similar affinities (K(d) ca. 200 nM). Moreover, in contrast to the mammalian enzyme, phosphoisoprenoids do not influence the affinity of Mrs6p for yRabGGTase. Using an in vitro prenylation assay, we have demonstrated that yRabGGTase can prenylate Rab proteins in complex with mammalian REP-1, thus indicating that neither the LRR nor the Ig-like domains, nor the recently discovered alternative pathway of catalytic complex assembly, are essential for the catalytic activity of RabGGTase. Despite the ability to function in concert with yRabGGTase in vitro, expression of mammalian REP-1 could not complement deletion of MRS6 gene in S. cerevisiae in vivo. The implications of these findings are discussed.     

A new motif for inhibitors of geranylgeranyl diphosphate synthase

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is believed to receive the substrate farnesyl diphosphate through one lipophilic channel and release the product geranylgeranyl diphosphate through another. Bisphosphonates with two isoprenoid chains positioned on the α-carbon have proven to be effective inhibitors of this enzyme. Now a new motif has been prepared with one isoprenoid chain on the α-carbon, a second included as a phosphonate ester, and the potential for a third at the α-carbon. The pivaloyloxymethyl prodrugs of several compounds based on this motif have been prepared and the resulting compounds have been tested for their ability to disrupt protein geranylgeranylation and induce cytotoxicity in myeloma cells. The initial biological studies reveal activity consistent with GGDPS inhibition, and demonstrate a structure–function relationship which is dependent on the nature of the alkyl group at the α-carbon.     

Synthesis and activity of p-azidobenzoyloxyferricrocin, a photoactivatable analog of ferrichrome

p-azidobenzoyloxy desferriferricrocin (AF) 2, a photoactivatable analog of ferrichrome, was prepared by selective acylation of the serine group of ferricrocin 1 in two steps: transesterification of ferricrocin followed by demetallation. A model compound, (L) 2-benzyloxycarbonylamino-3-p-azidobenzoyloxy N-isopropyl propionamide 8, was separately synthesized in order to set up optimal transesterification conditions to avoid , -elimination or epimerization of serine. Binding of iron-loaded AF (FeAF) to the FhuA outer membrane receptor protein of Escherichia coli AB2847 was demonstrated by inhibition of ferrichrome transport, interference with the infection by the bacteriophage 80 and with killing of cells by albomycin and colicin M. FeAF transported iron only weakly which indicates that the photoaffinity moiety is incompatible with transport or intracellular iron release from the siderophore.     

Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages

Nitrogen-containing bisphosphonate drugs inhibit bone resorption by inhibiting FPP synthase and thereby preventing the synthesis of isoprenoid lipids required for protein prenylation in bone-resorbing osteoclasts. NE10790 is a phosphonocarboxylate analogue of the potent bisphosphonate risedronate and is a weak anti-resorptive agent. Although NE10790 was a poor inhibitor of FPP synthase, it did inhibit prenylation in J774 macrophages and osteoclasts, but only of proteins of molecular mass approximately 22-26 kDa, the prenylation of which was not affected by peptidomimetic inhibitors of either farnesyl transferase (FTI-277) or geranylgeranyl transferase I (GGTI-298). These 22-26-kDa proteins were shown to be geranylgeranylated by labelling J774 cells with [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]mevalonic acid into Rab6, but not into H-Ras or Rap1, proteins that are modified by FTase and GGTase I, respectively. These data demonstrate that NE10790 selectively prevents Rab prenylation in intact cells. In accord, NE10790 inhibited the activity of recombinant Rab GGTase in vitro, but did not affect the activity of recombinant FTase or GGTase I. NE10790 therefore appears to be the first specific inhibitor of Rab GGTase to be identified. In contrast to risedronate, NE10790 inhibited bone resorption in vitro without markedly affecting osteoclast number or the F-actin "ring" structure in polarized osteoclasts. However, NE10790 did alter osteoclast morphology, causing the formation of large intracellular vacuoles and protrusion of the basolateral membrane into large, "domed" structures that lacked microvilli. The anti-resorptive activity of NE10790 is thus likely due to disruption of Rab-dependent intracellular membrane trafficking in osteoclasts.     

Interaction with the small subunit of geranyl diphosphate synthase modifies the chain length specificity of geranylgeranyl diphosphate synthase to produce geranyl diphosphate.

Geranyl diphosphate synthase belongs to a subgroup of prenyltransferases, including farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, that catalyzes the specific formation, from C(5) units, of the respective C(10), C(15), and C(20) precursors of monoterpenes, sesquiterpenes, and diterpenes. Unlike farnesyl diphosphate synthase and geranylgeranyl diphosphate synthase, which are homodimers, geranyl diphosphate synthase from Mentha is a heterotetramer in which the large subunit shares functional motifs and a high level of amino acid sequence identity (56-75%) with geranylgeranyl diphosphate synthases of plant origin. The small subunit, however, shares little sequence identity with other isoprenyl diphosphate synthases; yet it is absolutely required for geranyl diphosphate synthase catalysis. Coexpression in Escherichia coli of the Mentha geranyl diphosphate synthase small subunit with the phylogenetically distant geranylgeranyl diphosphate synthases from Taxus canadensis and Abies grandis yielded a functional hybrid heterodimer that generated geranyl diphosphate as product in each case. These results indicate that the geranyl diphosphate synthase small subunit is capable of modifying the chain length specificity of geranylgeranyl diphosphate synthase (but not, apparently, farnesyl diphosphate synthase) to favor the production of C(10) chains. Comparison of the kinetic behavior of the parent prenyltransferases with that of the hybrid enzyme revealed that the hybrid possesses characteristics of both geranyl diphosphate synthase and geranylgeranyl diphosphate synthase.     

Characterization of the ternary complex between Rab7, REP-1 and Rab geranylgeranyl transferase.

Geranylgeranylation is a post-translational modification of Rab GTPases that enables them to associate reversibly with intracellular membranes. Geranylgeranylation of Rab proteins is critical for their activity in controlling intracellular membrane transport. According to the currently accepted model for their action, newly synthesized Rab proteins are recruited by Rab escort protein (REP) and are presented to the Rab geranylgeranyl transferase (RabGGTase) which covalentely modifies the Rab protein with two geranylgeranyl moieties. After prenylation, the Rab protein remains in complex with REP and is delivered to the target membrane by the latter. In this work, we show that RabGGTase can form a stable complex with Rab7-REP in the absence of its lipid substrate geranylgeranyl pyrophosphate. In order to characterize this interaction, we developed three fluorescence assays reporting on the interaction of RabGGTase with the Rab7-REP complex. For this interaction we determined a Kd value of about 120 nM. Association of RabGGTase with the Rab7-REP complex occurs with a rate constant of approximately 108 M-1 x s-1. We demonstrate that the state of the nucleotide bound to Rab7 does not influence the affinity of RabGGTase for the Rab7-REP-1 complex. Finally, we address the issue of substrate specificity of RabGGTase. Titration experiments demonstrate that, in contrast with farnesyl transferase, RabGGTase does not recognize a defined C-terminal sequence motif. Experiments using Rab7 mutants in which the last 16 amino acids were either mutated or truncated revealed that the distal part of the C-terminus makes only a limited contribution to the binding affinity between RabGGTase and the Rab7-REP-1 complex. This demonstrates the functional dissimilarity between RabGGTase and geranylgeranyl transferase I and farnesyl transferase, which interact specifically with the C-terminus of their substrates. Based on these experiments, we propose that RabGGTase recognizes the overall structure arising from the association of Rab and REP and then 'scans' the flexible C-terminus to position the proximal cysteines into the active site.     

Inhibition of geranylgeranyl diphosphate synthesis in in vitro systems

The incorporation of [¹⁴C]mevalonate and [¹⁴C]isopentenyl diphosphate into geranylgeranyl diphosphate was investigated in in vitro systems from Cucurbita pepo (pumpkin) endosperm and from Avena sativa etioplasts. Mevalonate incorporation was effectively inhibited in the pumpkin system by geranylgeranyl diphosphate and geranylgeranyl monophosphate but less effectively by phytyl diphosphate or inorganic diphosphate. Membrane lipids, geranyllinalool, or lecithin enhanced mevalonate incorporation in the Cucurbita system. Incorporation of isopentenyl diphosphate was also enhanced by lecithin and inhibited by geranylgeranyl diphosphate in the Cucurbita system. No lipid enhancement was found in the Avena system; inhibition by GGPP required a much higher GGPP concentration than in the Cucurbita system.     

A versatile photoactivatable probe designed to label the diphosphate binding site of farnesyl diphosphate utilizing enzymes

Farnesyl diphosphate (FPP) is a substrate for a diverse number of enzymes found in nature. Photoactive analogues of isoprenoid diphosphates containing either benzophenone, diazotrifluoropropionate or azide groups have been useful for studying both the enzymes that synthesize FPP as well as those that employ FPP as a substrate. Here we describe the synthesis and properties of a new class of FPP analogues that links an unmodified farnesyl group to a diphosphate mimic containing a photoactive benzophenone moiety; thus, importantly, these compounds are photoactive FPP analogues that contain no modifications of the isoprenoid portion of the molecule that may interfere with substrate binding in the active site of an FPP utilizing enzyme. Two isomeric compounds containing meta- and para-substituted benzophenones were prepared. These two analogues inhibit Saccharomyces cerevisiae protein farnesyltransferase (ScPFTase) with IC₅₀ values of 5.8 (meta isomer) and 3.0 μM (para isomer); the more potent analogue, the para isomer, was shown to be a competitive inhibitor of ScPFTase with respect to FPP with a K_I of 0.46 μM. Radiolabeled forms of both analogues selectively labeled the β-subunit of ScPFTase. The para isomer was also shown to label Escherichia coli farnesyl diphosphate synthase and Drosophila melanogaster farnesyl diphosphate synthase. Finally, the para isomer was shown to be an alternative substrate for a sesquiterpene synthase from Nostoc sp. strain PCC7120, a cyanobacterial source; the compound also labeled the purified enzyme upon photolysis. Taken together, these results using a number of enzymes demonstrate that this new class of probes should be useful for a plethora of studies of FPP-utilizing enzymes.     

Structure and reaction geometry of geranylgeranyl diphosphate synthase from Sinapis alba

The crystal structure of the geranylgeranyl diphosphate synthase from Sinapis alba (mustard) has been solved in two crystal forms at 1.8 and 2.0 A resolutions. In one of these forms, the dimeric enzyme binds one molecule of the final product geranylgeranyl diphosphate in one subunit. The chainfold of the enzyme corresponds to that of other members of the farnesyl diphosphate synthase family. Whereas the binding modes of the two substrates dimethylallyl diphosphate and isopentenyl diphosphate at the allyl and isopentenyl sites, respectively, have been established with other members of the family, the complex structure presented reveals for the first time the binding mode of a reaction product at the isopentenyl site. The binding geometry of substrates and product in conjunction with the protein environment and the established chemistry of the reaction provide a clear picture of the reaction steps and atom displacements. Moreover, a comparison with a ligated homologous structure outlined an appreciable induced fit: helix alpha8 and its environment undergo a large conformational change when either the substrate dimethylallyl diphosphate or an analogue is bound to the allyl site; only a minor conformational change occurs when the other substrate isopentenyl diphosphate or the product is bound to the isopentenyl site.     

Structures of a potent phenylalkyl bisphosphonate inhibitor bound to farnesyl and geranylgeranyl diphosphate synthases

We report the X-ray crystallographic structures of the bisphosphonate N-[methyl(4-phenylbutyl)]-3-aminopropyl-1-hydroxy-1,1-bisphosphonate (BPH-210), a potent analog of pamidronate (Aredia), bound to farnesyl diphosphate synthase (FPPS) from Trypanosoma brucei as well as to geranylgeranyl diphosphate synthase from Saccharomyces cerevisiae. BPH-210 binds to FPPS, together with 3 Mg(2+), with its long, hydrophobic phenylbutyl side-chain being located in the same binding pocket that is occupied by allylic diphosphates and other bisphosphonates. Binding is overwhelmingly entropy driven, as determined by isothermal titration calorimetry. The structure is of interest since it explains the lack of potency of longer chain analogs against FPPS, since these would be expected to have a steric clash with an aromatic ring at the distal end of the binding site. Unlike shorter chain FPPS inhibitors, such as pamidronate, BPH-210 is also found to be a potent inhibitor of human geranylgeranyl diphosphate synthase. In this case, the bisphosphonate binds only to the GGPP product inhibitory site, with only 1 (chain A) or 0 (chain B) Mg(2+), and DeltaS is much smaller and DeltaH is approximately 6 k cal more negative than in the case of FPPS binding. Overall, these results are of general interest since they show that some bisphosphonates can bind to more than one trans-prenyl synthase enzyme which, in some cases, can be expected to enhance their overall activity in vitro and in vivo.     

Quantitative determination of farnesyl and geranylgeranyl diphosphate levels in mammalian tissue

Farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) are branch point intermediates of isoprenoid biosynthesis. Inhibitors of isoprenoid biosynthesis, such as the statins and bisphosphonates, are widely used therapeutic agents. However, little is known about the degree to which they alter levels of upstream and downstream isoprenoids, including FPP and GGPP. Therefore, we developed a method to isolate and quantify FPP and GGPP from mammalian tissues. Tissues from mice were collected, snap frozen in liquid nitrogen, and stored at −80 °C. FPP and GGPP were isolated by a combined homogenization and extraction procedure and were purified with a C18 solid phase extraction column. Farnesyl protein transferase (FTase) or geranylgeranyl protein transferase I (GGTase I) were used to conjugate FPP and GGPP with fluorescent dansylated peptides. FPP and GGPP were quantified by high-performance liquid chromatography (HPLC). The respective concentrations of FPP and GGPP are as follows: 0.355 ± 0.030 and 0.827 ± 0.082 units of nmol/g wet tissues in brain, 0.320 ± 0.019 and 0.293 ± 0.035 units of nmol/g wet tissues in kidney, 0.326 ± 0.064 and 0.213 ± 0.029 units of nmol/g wet tissues in liver, and 0.364 ± 0.015 and 0.349 ± 0.023 units of nmol/g wet tissues in heart (means ± SEM). This method allows for determination of FPP and GGPP concentrations in any tissue type and is sensitive enough to detect changes following treatment with inhibitors of isoprenoid biosynthesis.