FPP/farnesyl pyrophosphate synthase

The incorporation of [14C]mevalonate into Rab6 is inhibited by NE 10790, but not into H-Ras or Rap1 (proteins that have been altered by FTase and GGTase I, respectively). J774 cells have less viability when exposed to NE 10790. Prenylation of 22–26 kDa proteins that are not altered by FTase or GGTase I is inhibited by NE 10790 [1].

NE10790 is an analogue of the nitrogen-containing BP RIS, in which one of the phosphonate groups is replaced with a carboxylate group. NE10790 retains the ability to inhibit bone resorption in vivo, although its anti-resorptive potency in rodents is markedly reduced compared with RIS. At least part of this loss of potency is due to the fact that NE10790 has reduced affinity for bone, because the loss of one of the phosphonate groups allows binding of only one calcium ion. However, it remains unclear whether this compound is also less effective at affecting osteoclast function at the cellular level or indeed whether it inhibits bone resorption by the same molecular mechanism as nitrogen-containing BPs (that is, by inhibition of FPP synthase). NE10485 is an analogue of NE10790 in which the nitrogen of the heterocyclic group is methylated and the hydroxyl group attached to the central carbon is replaced with hydrogen. The anti-resorptive potency of this compound has not been characterized[1].

FPP Synthase Assay[1]
FPP synthase was assayed as described previously. Briefly, 40 μl of assay buffer (50 mm Tris, pH 7.7, 10 mm NaF, 2 mmMgCl2, 1 mg/ml bovine serum albumin, 0.5 mmdithiothreitol) containing 2 nmol [1-14C]isopentenyl diphosphate (4 μCi/mmol) and 2 nmol GPP were prewarmed to 37 °C. The assay was initiated by the addition of 1 μl of recombinant human FPP synthase (with an activity of 8 pmol FPP/min) diluted to 10 μl with assay buffer. The assay was allowed to proceed for 30 min and was terminated by the addition of 200 μl of saturated NaCl. The samples were then extracted with 1 ml of water-saturated butan-1-ol, and the amount of radioactivity in the upper phase was determined by mixing 0.5 ml of the butyl alcohol with 4 ml of general purpose scintillant. This was then counted using a Packard Tricarb 1900CA scintillation counter. To determine the effects of NE10790, RIS, and NE10485 on FPP synthase activity, the compounds were diluted to 5× final concentration in assay buffer and were preincubated with the enzyme preparation for 10 min prior to initiation of the reaction.

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Protein:Farnesyl Transferase Assay[1]
The activity of recombinant human farnesyl transferase was determined by assessing the amount of [3H]farnesyl transferred from [3H]FPP to recombinant K-Ras (22). The final concentrations of reagents in a standard reaction mix were 50 mm Tris-Cl, pH 7.2, 150 mm KCl, 10 mm ZnCl2, 3 mm MgCl2, 0.2% (v/v) octyl-d-glucopyranoside (NOGA detergent), 1 mm dithiothreitol, 0.9 μm FPP, 0.3 μm [3H]FPP (15–30 Ci/mmol), 40 nm FTase, and 15 μm K-Ras. NE10790, NE10485, and RIS were aliquoted into siliconized microcentrifuge tubes at 4 °C and added to the reaction mix. The reactions were initiated by transferring the tubes to 37 °C and incubated for 15 min, and then reactions were stopped by the addition of 400 μl of ethanol:HCl (9:1 v/v). The protein was allowed to precipitate at room temperature for 30 min and was then filtered on to glass microfiber filters (GF/C). The tubes were washed three times with 1 ml of ethanol, and then the amount of radioactivity on each filter quantified by liquid scintillation counting following the addition of 5 ml of scintillation mixture. The assays were in duplicate and were repeated three times independently to verify reproducibility.

Assessment of Viable Cell Number by MTT Assay[1]
The number of viable J774 macrophage cells was determined by MTT assay as previously described. J774 cells were seeded at a density of 104cells/well into 96-well plates and then treated with RIS, NE10790, or NE10485 the following day in replicates of six wells. 48 h later, the reduction of MTT reagent was measured.

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Incorporation of [14C]Mevalonate and [3H]GGOH into Prenylated Proteins in Intact Cells[1]
Detection of prenylated proteins in J774 macrophages and purified rabbit osteoclasts was carried out as described previously (16, 24). Briefly, the cells were depleted of mevalonate by incubation with 5 μm mevastatin for 4 h and then transferred into fresh medium containing 5 μm mevastatin and either 7.5 μCi/ml [14C]mevalonic acid lactone or 30 μCi/ml [3H]GGOH, plus RIS, NE10790, NE10485, FTI-277, or GGTI-298. After 18 h the cells were lysed in RIPA buffer (1% (v/v) Nonidet P-40, 0.1% (w/v) sodium dodecyl sulfate, 0.5% (w/v) sodium deoxycholate in PBS, plus 1:100 (v/v) Sigma protease inhibitor mixture), and then 50 μg of cell lysate from each treatment were electrophoresed on 12% polyacrylamide-SDS gels under reducing conditions. After electrophoresis, the gels were fixed in 10% (v/v) acetic acid, 40% (v/v) methanol, 50% (v/v) distilled water, and then14C-labeled gels were dried, and labeled proteins were visualized on a Bio-Rad Personal FX Imager after exposure to a Kodak phosphorimaging screen. 3H-Labeled gels were incubated in Enhance for 30 min prior to drying. 3H-Labeled proteins were then visualized by exposing the gel to preflashed Hyperfilm-MP for 6 days at −70 °C.

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Analysis of Osteoclast Polarization and Resorption[1]
Osteoclast number, F-actin “rings,” and resorptive activity of mature rabbit osteoclasts in vitro were assessed as described previously. Briefly, rabbit osteoclasts were allowed to adhere for 2 h on 5-mm-diameter elephant tusk dentine discs in 96-well plates and then cultured with fresh α-minimum essential medium in the presence or absence of RIS, NE10790, or NE10485. 48 h later, intracellular F-actin was visualized by staining with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin, and the number of actin rings, defined as a distinct and complete ring of podosomes, per disc was then counted (actin rings are an indication of osteoclast polarization, and their presence correlates highly with active resorption). The discs were then stained for tartrate-resistant acid phosphatase by incubating with naphthol-ASBI-phosphate, hexazotized pararosanilin, and 50 mm tartrate in acetate buffer, pH 5.5, at 37 °C for 30 min. To detect the total number of osteoclasts in the cultures, the number of multinucleated (>2 nuclei/cell), tartrate-resistant acid phosphatase-positive cells/disc was then counted. To assess osteoclastic resorption, the discs were immersed in 20% (w/v) sodium hypochlorite to remove all cells, and then resorption pits in the mineral surface were visualized by reflected light microscopy. The area of the pits/disc was then examined using a Zeiss Axiolab reflective light microscope and quantified using software developed in-house based on Aphelion ActiveX components.

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Assessment of Osteoclast Morphology by Immunostaining and Confocal Microscopy[1]
For morphological studies, rabbit osteoclasts were cultured as above for 48 h on dentine slices without reagents or with 10–100 μm RIS or 500–1000 μmNE10790. The cultures were fixed for 10 min in a 1:1 (v:v) mixture of minimum essential medium and fixation buffer (3.5% (w/v) paraformaldehyde, 2% (w/v) sucrose in PBS). The cells were then permeablized in Triton buffer (20 mm Hepes, 300 mm sucrose, 50 mm NaCl, 3 mmMgCl2, 0.5% (v/v) Triton X-100, 0.5% (w/v) sodium azide, in PBS at pH 7.0) at 4 °C and then immunostained for paxillin using a mouse monoclonal antibody (10 μg/ml) and secondary fluorescein isothiocyanate-conjugated anti-mouse Ig polyclonal antibodies (1:40 dilution). Resorbing osteoclasts were identified by their characteristic F-actin ring structure (described above) after staining with TRITC-phalloidin conjugate (Molecular Probes) at 5 units/ml. The dentine surface was examined by simultaneous confocal reflection microscopy where resorbed areas could be identified by their reduced reflection. Scanning laser confocal microscopy was performed on a Leica TCS NT system. Fluorescent images were collected in sequential 1-μm steps through the osteoclasts for fluorescein isothiocyanate and TRITC fluorochromes and reflection at 488-, 568-, and 647-nm emission wavelengths, respectively, and displayed in the xy and zx planes.

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Analysis of Osteoclast Morphology by Transmission Electron Microscopy[1]
For electron microscopic analysis, rabbit osteoclasts were treated with or without 1 mm NE10790 for 48 h on dentine slices. The cells were fixed in 2.5% (v/v) glutaraldehyde in phosphate buffer (0.1 m, pH 7.4) for a minimum of 24 h and then demineralized in the same fixative with 2.5% (w/v) EDTA for ∼48 h. The samples were then washed in buffer, postfixed in osmium tetroxide, dehydrated in ethanol, and embedded in Epon. Ultrathin sections were stained using uranyl acetate and lead citrate and then examined using a Philips EM201 microscope.

[1]. Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages. J Biol Chem. 2001 Dec 21;276(51):48213-22.

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.[1]

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To our knowledge, NE10790 is the first specific inhibitor of Rab prenylation to be identified. Other inhibitors of Rab GGTase, such as the monoterpene perillyl alcohol, are less specific and also affect GGTase I and FTase. NE10790 will therefore be a useful tool for further characterization of the role that Rab proteins play in cellular processes necessary for resorption by osteoclasts. Furthermore, the ability of NE10790 to specifically inhibit Rab prenylation in J774 macrophages, osteoclasts, and MC3T3 osteoblast-like cells suggests that this compound will be useful for examining the role of Rab proteins in a variety of cell types in vitro. Until now, the only insights into the consequences of loss of Rab prenylation have come from studies on the effect of genetic mutations in the Rab prenylation machinery. For example, a mutation in REP1 leads to a retinal degenerative disease called choroideremia. The loss of REP1 is partially compensated by the homologous REP2 protein but results in a selective defect in prenylation of a subset of Rab proteins. In addition, mutations in the yeast REP (mrs6-2) results in reduced prenylation of Rab proteins, causing failure of the polarized transport of vesicles toward the bud. Finally, mice with a mutation known asgunmetal have a 4-fold decrease in Rab GGTase activity in platelets as a result of a single base substitution that disrupts the splicing of the Rabggta mRNA. These mice exhibit prolonged bleeding, thrombocytopaenia, and reduced platelet contents, suggesting that Rab GGTase could be a novel target for the treatment of clotting disorders such as myocardial infarction and stroke.
The mechanism by which NE10790 inhibits Rab GGTase remains to be determined. Because the closely related analogue NE10485 (which differs only in that the nitrogen in the side chain is methylated and the hydroxyl group attached to the geminal carbon atom is replaced by a hydrogen) did not inhibit Rab GGTase and had no effect on protein prenylation, the use of other phosphonocarboxylate analogues similar to NE10790 may help to shed light on the molecular structures necessary for interaction with Rab GGTase. Furthermore, although the low potency of NE10790 could limit its potential therapeutic use, future studies addressing the inhibitory mechanism of NE10790 will ultimately help in the rational design of new, more potent inhibitors of this Rab GGTase that have potential for the treatment of thrombotic disorders in addition to diseases of excessive bone resorption such as post-menopausal osteoporosis.
In summary, we demonstrate in this study that NE10790, a phosphonocarboxylate analogue of the potent anti-resorptive drug RIS, inhibits Rab GGTase in vitro and specifically prevents the prenylation of Rab GTPases. NE10790 is therefore a useful new tool that provides a novel approach for investigating the function of Rab proteins in osteoclasts and other cell types.[1]

C8H10NO6P

247.1419

247.025

C, 38.88; H, 4.08; N, 5.67; O, 38.84; P, 12.53

152831-36-2

10014835

White to off-white solid powder

-1.8

4

7

4

16

316

0

FJVYPXVLXQXDHM-UHFFFAOYSA-N

InChI=1S/C8H10NO6P/c10-7(11)8(12,16(13,14)15)4-6-2-1-3-9-5-6/h1-3,5,12H,4H2,(H,10,11)(H2,13,14,15)

2-hydroxy-2-phosphono-3-(pyridin-3-yl)propanoic acid

3-PEHPC; 3 PEHPC; 3PEHPC; NE10790; 2-hydroxy-2-phosphono-3-(pyridin-3-yl)propanoic acid; 3Pehpc; 2-hydroxy-2-phosphono-3-pyridin-3-ylpropanoic acid; NE 10790; NE-10790.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

0.1 M NaOH : ~20 mg/mL (~80.93 mM)
H2O : ~5 mg/mL (~20.23 mM)

Solubility in Formulation 1: 2 mg/mL (8.09 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)