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Identification of a Bisphosphonate That Inhibits Isopentenyl Diphosphate Isomerase and Farnesyl Diphosphate Synthase
Biochemical and Biophysical Research Communications 290, 869 – 873 (2002) doi:10.1006/bbrc.2001.6289, available online at http://www.idealibrary.com on
Identification of a Bisphosphonate That Inhibits Isopentenyl Diphosphate Isomerase and Farnesyl Diphosphate Synthase Keith Thompson,* James E. Dunford,* Frank H. Ebetino,† and Michael J. Rogers* ,1 *Bone Research Group, Department of Medicine & Therapeutics, University of Aberdeen, Scotland, United Kingdom; and †Proctor & Gamble Pharmaceuticals, Health Care Research Center, Mason, Ohio 45040
Received December 6, 2001
We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC 50 values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase. © 2002 Elsevier Science
Key Words: bisphosphonate; farnesyl diphosphate synthase; isopentenyl diphosphate isomerase; mevalonate; prenylation; osteoclast.
Bisphosphonates are commonly used drugs to inhibit excessive osteoclast-mediated bone resorption in patients with osteoporosis, metastatic bone disease and hypercalcemia (1, 2). Bisphosphonates inhibit osteoclast formation in vitro, disrupt the osteoclast cytoskeleton and ruffled border, and can cause osteoclast apoptosis, reviewed in Ref. (3). We and others have recently demonstrated that bisphosphonates that con1
To whom correspondence and reprint requests should be addressed at Bone Research Group, Department of Medicine & Therapeutics, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, UK. Fax: 44 1224 699884. E-mail: [email protected].
tain a nitrogen in one of their two side-chains (N-BPs, such as alendronate, risedronate, ibandronate, and pamidronate) act by inhibiting the mevalonate pathway and, therefore, indirectly prevent the posttranslational prenylation of small GTPases such as Rho, Rac, and cdc42 (4 – 8). By preventing the prenylation of small GTPases in osteoclasts, N-BPs affect signal transduction pathways regulating cytoskeletal arrangement, membrane ruffling, vesicular trafficking and apoptosis (9 –11). Inhibition of protein prenylation therefore accounts for the effects of N-BPs on osteoclast morphology, function and survival. The enzyme of the mevalonate pathway that is the target of N-BPs has recently been identified as farnesyl diphosphate (FPP) synthase (7, 12). N-BPs are potent inhibitors of FPP synthase and there is a highly significant correlation between the rank order of potency for inhibition of FPP synthase in vitro and inhibition of bone resorption in vivo (13). Although FPP synthase appears to be the major pharmacological target of N-BPs, we have examined whether these compounds may also inhibit other enzymes in the mevalonate pathway, such as isopentenyl diphosphate (IPP) isomerase, the enzyme proximal to FPP synthase in the mevalonate pathway (Fig. 1). In this study we describe a novel N-BP, NE21650, that inhibits both FPP synthase and IPP isomerase, and NE10571, a pharmacologically inactive isomer of NE21650. MATERIALS AND METHODS Reagents. The sodium salts of alendronate (ALN; 4-aminobutane-1,1-bisphosphonic acid), NE21650 (2-aminophenyl-2-ethane1-hydroxy-1,1-bisphosphonic acid) and NE10571 (4-aminophenyl-2ethane-1,1-bisphosphonic acid) were synthesized by Proctor & Gamble Pharmaceuticals (Cincinnati, OH). The N-BPs were dissolved in PBS, the pH adjusted to 7.4 with 1 N NaOH, and then filtersterilized using a 0.2-m filter. All other reagents were obtained from Sigma Chemical Co., unless otherwise stated. [ 14C]Mevalonic acid lactone was purchased from NEN (Hounslow, UK).
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Expression of recombinant human IPP isomerase and FPP synthase. The human IPP isomerase clone pFMH12 in the bacterial expression vector pARC306N (14) was kindly provided by C. D. Poulter, University of Utah and expressed as previously described (13). Human FPP synthase (Clone KIA1293, a kind gift from the Kazusa DNA Research Institute, Kisarazu, Chiba, Japan) was expressed and partially purified as described previously (13). In vitro assay of IPP isomerase and FPP synthase activity. IPP isomerase activity was assayed by the acid-lability method (15) using [ 14C]IPP as a substrate, as previously described (13). The hydrolyzed reaction products were solvent extracted into ligroin and the radioactivity in the ligroin fraction (dimethylallyl diphosphate, the product of the isomerase reaction) was determined using a Packard Tricarb 1900CA scintillation counter. N-BPs were pre-incubated with the enzyme for 10 min prior to initiation of the reaction. FPP synthase activity was assayed using [ 14C]IPP and geranyl diphosphate as substrates, as described previously (13). Reaction products were extracted with water-saturated butyl alcohol and the amount of radioactivity in the upper phase was determined by scintillation counting, using a Packard Tricarb 1900CA scintillation counter. To determine the effects of N-BPs on FPP synthase activity, N-BPs were pre-incubated with the enzyme preparation for 10 min, prior to initiation of the reaction. Effect of N-BPs on Rap1A prenylation in osteoclasts and macrophages. Osteoclasts were isolated from the long bones of 2-day-old New Zealand white rabbits, seeded into 6-well culture plates then purified according to the method of Coxon et al. (10). J774 cells were cultured in 12-well plates (1 ⫻ 10 5 cells/well). After treatment with 50 M N-BPs for 24 h, J774 cells and osteoclasts were lysed in 0.1 ml 1% (v/v) NP-40, 0.1% (w/v) sodium dodecyl sulfate, 0.5% (w/v) sodium deoxycholate, 1% (v/v) protease inhibitor cocktail (Sigma). Twenty micrograms of J774 cell lysate or 50 g of rabbit osteoclast lysate was then electrophoresed on 12% polyacrylamide–SDS gels under reducing conditions. Following electrophoresis, the proteins were transferred to polyvinyldifluoride (PVDF) membrane and then hybridized with 0.2 g/ml goat polyclonal anti-Rap1A antibody (Santa Cruz Biotechnology Inc.), which recognizes only the unprenylated form of Rap1A (16, 17) followed by 1 g/ml anti-goat IgG– horseradish peroxidase (HRP) conjugate (Sigma). Chemiluminescent bands were visualized using Supersignal reagent (Pierce) and a Bio-Rad Fluor-S Max MultiImager. Incorporation of [ 14C]mevalonate into prenylated proteins in macrophages. Protein prenylation in J774 macrophages was studied by metabolically labeling cells with [ 14C]mevalonic acid lactone for 20 h in the presence of 50 or 100 M N-BPs, as described previously (4). Cell lysates were then examined for the presence of radiolabeled, prenylated proteins. Fifty micrograms of protein from each cell lysate was separated on 12% polyacrylamide–SDS gels under reducing conditions, then prenylated proteins were visualized by phosphorimaging, using a Kodak phosphorscreen and Bio-Rad Personal FX imager. Effects of N-BPs on osteoclast-mediated bone resorption. Rabbit osteoclasts were seeded on to 5-mm-diameter elephant tusk ivory slices in 96-well plates and allowed to adhere for 2 h. Following 48 h exposure with 0.1–100 M N-BPs, the slices were fixed with 4% formaldehyde and resorptive activity of osteoclasts was assessed by reflected light microscopy using a Leitz Quantimet Q500MC image analysis system, as described by van’t Hof et al. (18).
RESULTS AND DISCUSSION In this study we screened N-BPs for the ability to inhibit FPP synthase and IPP isomerase. NE21650, a bisphosphonate containing an amino group in the ortho-position of an aromatic ring (Fig. 1), was a potent
FIG. 1. Schematic representation of the mevalonate pathway, and the structures of nitrogen-containing bisphosphonates.
inhibitor of recombinant human FPP synthase, comparable in potency to ALN (IC 50 58 and 53 nM, respectively; Fig. 2). However, unlike ALN (Fig. 2) and other N-BPs studied previously, such as ibandronate, risedronate and incadronate (13), NE21650 also inhibited recombinant human IPP isomerase in vitro (IC 50 ⬃70 M; Fig. 2). NE10571 (an isomer of NE21650 containing a para-amino group rather than an ortho-amino group), had no inhibitory effect on IPP isomerase at concentrations up to 300 M, and was almost 700-fold less potent than NE21650 at inhibiting recombinant human FPP synthase. This indicates that the position of the amino group in an aromatic ring is an important determinant for inhibition of IPP isomerase and FPP synthase by bisphosphonates. This is in accord with our recent observation that the three-dimensional position of a nitrogen within a heterocyclic ring is critical for effective inhibition of FPP synthase by bisphosphonates (13). We next investigated whether, by inhibiting both FPP synthase and IPP isomerase, NE21650 was a more effective inhibitor of protein prenylation in vitro than ALN (which inhibits FPP synthase only, with a very similar IC 50 to NE21650). Western blot analysis, using an antibody that selectively recognizes the unprenylated form of Rap1A (16, 17), showed that NE21650 was a more effective inhibitor of protein pre-
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FIG. 3. Inhibition of prenylation of Rap1A by ALN and NE21650, but not NE10571, in J774 macrophages (A) and purified rabbit osteoclasts (B). Cells were treated with 50 M N-BPs for 24 h prior to separation of proteins in cell lysates by SDS–PAGE, then Western blotting using an antibody that recognizes the 21-kDa, unprenylated form of Rap1A (arrowheads). Blots were also probed with anti-actin antibody to ensure equal loading per lane (not shown). Data are representative of three independent experiments.
FIG. 2. (A) Inhibition of recombinant human IPP isomerase by NE21650, but not NE10571 or ALN. N-BPs were incubated with recombinant human IPP isomerase for 10 min prior to assaying activity. [ 14C]DMAPP formed in the reaction was extracted into ligroin and the activity determined by liquid scintillation counting. Data are the mean ⫾ SEM of at least three independent experiments, expressed as a percentage of IPP isomerase activity in the absence of N-BP. (B) Inhibition of recombinant human FPP synthase by NE21650 and ALN. N-BPs were incubated with recombinant human FPP synthase for 10 min prior to assaying for FPP synthase activity. 14C-labeled FPP and geranyl diphosphate formed in the reaction were extracted into water-saturated butanol and activity determined by liquid scintillation counting. Data are means ⫾ SEM of at least three independent experiments, expressed as a percentage of FPP synthase activity in the absence of N-BP.
nylation than ALN in both J774 macrophages and purified rabbit osteoclasts, since unprenylated Rap1A accumulated to a greater extent in cells treated with NE21650 compared to cells treated with ALN (Fig. 3). This suggests that inhibition of both IPP isomerase and FPP synthase prevents protein prenylation more effectively in intact cells than inhibition of FPP synthase alone. NE10571 did not cause accumulation of unprenylated Rap1A in J774 cells (Fig. 3), consistent with the lack of effect of this compound on IPP isomerase or FPP synthase. The greater effectiveness of NE21650 for inhibiting protein prenylation was confirmed by labeling J774 macrophages with [ 14C]mevalonate. At a concentration of 50 M, NE21650 clearly prevented the synthesis of isoprenoid lipids, that migrate at the dye front of SDS– PAGE gels (4, 10), but only slightly prevented the
prenylation of 21- to 26-kDa small GTPases (Fig. 4). ALN (50 M) had little effect on the synthesis of isoprenoid lipids or on protein prenylation. At a concentration of 100 M, NE21650 completely prevented the synthesis of isoprenoid lipids and very markedly prevented prenylation of small GTPases, whereas ALN was less effective. As expected, NE10571 had no effect on the synthesis of isoprenoid lipids or on protein prenylation (Fig. 4). Finally, given the apparent increase in the effectiveness of NE21650 for inhibiting protein prenylation, we examined whether this would confer an increase in antiresorptive potency compared to ALN. In an in vitro bone resorption assay using rabbit osteoclasts, NE21650 was slightly (but significantly) more effective than ALN at inhibiting resorption at concentrations ⱖ10 M (Fig. 5). Hence, NE21650, by inhibiting both
FIG. 4. Inhibition of protein prenylation by ALN and NE21650, but not NE10571, in J774 macrophages. Cells were metabolically labeled for 20 h with [ 14C]mevalonate in the absence or presence of 50 or 100 M ALN, NE10571, or NE21650. Cell lysates were analyzed by SDS–PAGE on 12% polyacrylamide gels and radiolabeled, prenylated proteins were detected by phosphorimaging. The position of isoprenoid intermediates and 21- to 26-kDa, prenylated small GTPases are indicated.
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ACKNOWLEDGMENTS We are grateful to C. D. Poulter and F. M. Hahn for providing the cDNA clone for IPP isomerase.
REFERENCES
FIG. 5. Inhibition of osteoclastic bone resorption by N-BPs in rabbit osteoclasts. Cells were treated with 0.1–100 M N-BPs for 48 h prior to quantification of the area of resorbed mineral by reflected light microscopy. Data are means ⫾ SEM of at least three independent experiments, expressed as a percentage of the area of mineral resorbed in untreated cultures.
IPP isomerase and FPP synthase, is more effective than ALN at inhibiting protein prenylation in osteoclasts and, consequently, has slightly increased antiresorptive potency. NE10571 did not inhibit bone resorption at all concentrations studied (up to 100 M). This study emphasizes the critical importance of the three-dimensional position of the nitrogen group in the bisphosphonate side chain (for example, ortho- vs paraposition in an aromatic ring) for determining the ability to inhibit FPP synthase and hence for determining the potency for inhibiting bone resorption. Furthermore, our findings illustrate that certain side-chain structures, such as NE21650, also allow the inhibition of other enzymes in the mevalonate pathway, such as IPP isomerase. The N-BPs ibandronate and incadronate, as well as inhibiting FPP synthase, are also known to inhibit squalene synthase (19, 20), thereby preventing cholesterol biosynthesis (Fig. 1). However, inhibition of squalene synthase does not prevent protein prenylation, and replenishing osteoclasts with cholesterol does not overcome the effects of N-BPs (5). Furthermore, the antiresorptive potency of ibandronate and incadronate correlates well with their ability to inhibit FPP synthase alone (13). Hence, inhibition of squalene synthase does not appear to directly enhance the antiresorptive effect of ibandronate or incadronate. By contrast, inhibition of IPP isomerase synthase by NE21650 appears to enhance the ability to prevent protein prenylation in intact cells and hence leads to a slight increase in antiresorptive potency compared to bisphosphonates that prevent protein prenylation by inhibiting only FPP synthase. The exact threedimensional structure of NE21650 that allows inhibition of IPP isomerase and FPP synthase remains to be determined.
1. Russell, G., Mueller, G., Shipman, C., and Croucher, P. (2001) Clinical disorders of bone resorption. Novartis Found. Symp. 232, 251–267. 2. Watts, N. B. (2001) Treatment of osteoporosis with bisphosphonates. Rheum. Dis. Clin. North Am. 27, 197–214. 3. Rogers, M. J., Gordon, S., Benford, H. L., Coxon, F. P., Luckman, S. P., Monkkonen, J., and Frith, J. C. (2000) Cellular and molecular mechanisms of action of bisphosphonates. Cancer 88, 2961–2978. 4. Luckman, S. P., Hughes, D. E., Coxon, F. P., Russell, R. G. G., and Rogers, M. J. (1998) Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras. J. Bone Miner. Res. 13, 581–589. 5. Fisher, J., Rogers, M. J., Halasy, J. M., Luckman, S. P., Hughes, D. E., Masarachia, P. J., Wesolowski, G., Russell, R. G. G., Rodan, G. A., and Reszka, A. A. (1999) Alendronate mechanism of action: Geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption and kinase activation in vitro. Proc. Natl. Acad. Sci. USA 96, 133–138. 6. van Beek, E., Lowik, C., Van der Pluijm, G., and Papapoulos, S. (1999) The role of geranylgeranylation in bone resorption and its suppression by bisphosphonates in fetal bone explants in vitro: A clue to the mechanism of action of nitrogen-containing bisphosphonates. J. Bone Miner. Res. 14, 722–729. 7. Bergstrom, J. D., Bostedor, R. G., Masarachia, P. J., Reszka, A. A., and Rodan, G. (2000) Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch. Biochem. Biophys. 373, 231–241. 8. Benford, H. L., Frith, J. C., Auriola, S., Monkkonen, J., and Rogers, M. J. (1999) Farnesol and geranylgeraniol prevent activation of caspases by aminobisphosphonates: Biochemical evidence for two distinct pharmacological classes of bisphosphonate drugs. Mol. Pharmacol. 56, 131–140. 9. Reszka, A. A., Halasy-Nagy, J. M., Masarachia, P. J., and Rodan, G. A. (1999) Bisphosphonates act directly on the osteoclast to induce caspase cleavage of mst1 kinase during apoptosis. A link between inhibition of the mevalonate pathway and regulation of an apoptosis-promoting kinase. J. Biol. Chem. 274, 34967– 34973. 10. Coxon, F. P., Helfrich, M. H., van ‘t Hof, R. J., Sebti, S. M., Ralston, S. H., Hamilton, A. D., and Rogers, M. J. (2000) Protein geranylgeranylation is required for osteoclast formation, function, and survival: Inhibition by bisphosphonates and GGTI-298. J. Bone Miner. Res. 15, 1467–1476. 11. Benford, H. L., McGowan, N. W., Helfrich, M. H., Nuttall, M. E., and Rogers, M. J. (2001) Visualization of bisphosphonateinduced caspase-3 activity in apoptotic osteoclasts in vitro. Bone 28, 465– 473. 12. van Beek, E., Pieterman, E., Cohen, L., Lowik, C., and Papapoulos, S. (1999) Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates. Biochem. Biophys. Res Commun. 264, 108 –111. 13. Dunford, J. E., Thompson, K., Coxon, F. P., Luckman, S. P., Hahn, F. M., Poulter, C. D., Ebetino, F. H., and Rogers, M. J. (2001) Structure–activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption
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in vivo by nitrogen-containing bisphosphonates. J. Pharmacol. Exp. Ther., 235–242. Hahn, F. M., Xuan, J. W., Chambers, A. F., and Poulter, C. D. (1996) Human isopentenyl diphosphate:dimethylallyl diphosphate isomerase: Overproduction, purification, and characterization. Arch. Biochem. Biophys. 332, 30 –34. Satterwhite, D. M. (1985) Isopentenyldiphosphate deltaisomerase. Methods Enzymol. 110, 92–99. Reszka, A. A., Halasy-Nagy, J., and Rodan, G. A. (2001) Nitrogen-bisphosphonates block retinoblastoma phosphorylation and cell growth by inhibiting the cholesterol biosynthetic pathway in a keratinocyte model for esophageal irritation. Mol. Pharmacol. 59, 193–202. Frith, J. C., Monkkonen, J., Auriola, S., Monkkonen, H., and Rogers, M. J. (2001) The molecular mechanism of action of the anti-resorptive and anti-inflammatory drug clodronate: Evi-
dence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis. Arthritis Rheum. 44, 2201–2210. 18. Van’t Hof, R. J., Tuinenburg-Bol, R. A., and Nijweide, P. J. (1995) Induction of osteoclast characteristics in cultured avian blood monocytes: Modulation by osteoblasts and 1,25-(OH)2 vitamin D3. Int. J. Exp. Pathol. 76, 205–214. 19. Amin, D., Cornell, S. A., Gustafson, S. K., Needle, S. J., Ullrich, J. W., Bilder, G. E., and Perrone, M. H. (1992) Bisphosphonates used for the treatment of bone disorders inhibit squalene synthase and cholesterol biosynthesis. J. Lipid Res. 33, 1657– 1663. 20. Amin, D., Cornell, S. A., Perrone, M. H., and Bilder, G. E. (1996) 1-Hydroxy-3-(methylpentylamino)-propylidene-1,1-bisphosphonic acid as a potent inhibitor of squalene synthase. ArzneimittelForschung. 46, 759 –762.
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Identification of a Bisphosphonate That Inhibits Isopentenyl Diphosphate Isomerase and Farnesyl Diphosphate Synthase Keith Thompson,* James E. Dunford,* Frank H. Ebetino,† and Michael J. Rogers* ,1 *Bone Research Group, Department of Medicine & Therapeutics, University of Aberdeen, Scotland, United Kingdom; and †Proctor & Gamble Pharmaceuticals, Health Care Research Center, Mason, Ohio 45040
Received December 6, 2001
We and others have recently shown that the major molecular target of nitrogen-containing bisphosphonate drugs is farnesyl diphosphate synthase, an enzyme in the mevalonate pathway. In an in vitro screen, we discovered a bisphosphonate, NE21650, that potently inhibited farnesyl diphosphate synthase but, unlike other N-BPs investigated, was also a weak inhibitor of isopentenyl diphosphate isomerase. NE21650 was a more potent inhibitor of protein prenylation in osteoclasts and macrophages, and a more potent inhibitor of bone resorption in vitro, than alendronate, despite very similar IC 50 values for inhibition of farnesyl diphosphate synthase. Our observations show that minor changes to the structure of bisphosphonates allow inhibition of more than one enzyme in the mevalonate pathway and suggest that loss of protein prenylation due to inhibition of more than one enzyme in the mevalonate pathway may lead to an increase in antiresorptive potency compared to bisphosphonates that only inhibit farnesyl diphosphate synthase. © 2002 Elsevier Science
Key Words: bisphosphonate; farnesyl diphosphate synthase; isopentenyl diphosphate isomerase; mevalonate; prenylation; osteoclast.
Bisphosphonates are commonly used drugs to inhibit excessive osteoclast-mediated bone resorption in patients with osteoporosis, metastatic bone disease and hypercalcemia (1, 2). Bisphosphonates inhibit osteoclast formation in vitro, disrupt the osteoclast cytoskeleton and ruffled border, and can cause osteoclast apoptosis, reviewed in Ref. (3). We and others have recently demonstrated that bisphosphonates that con1
To whom correspondence and reprint requests should be addressed at Bone Research Group, Department of Medicine & Therapeutics, University of Aberdeen, Polwarth Building, Foresterhill, Aberdeen AB25 2ZD, UK. Fax: 44 1224 699884. E-mail: [email protected].
tain a nitrogen in one of their two side-chains (N-BPs, such as alendronate, risedronate, ibandronate, and pamidronate) act by inhibiting the mevalonate pathway and, therefore, indirectly prevent the posttranslational prenylation of small GTPases such as Rho, Rac, and cdc42 (4 – 8). By preventing the prenylation of small GTPases in osteoclasts, N-BPs affect signal transduction pathways regulating cytoskeletal arrangement, membrane ruffling, vesicular trafficking and apoptosis (9 –11). Inhibition of protein prenylation therefore accounts for the effects of N-BPs on osteoclast morphology, function and survival. The enzyme of the mevalonate pathway that is the target of N-BPs has recently been identified as farnesyl diphosphate (FPP) synthase (7, 12). N-BPs are potent inhibitors of FPP synthase and there is a highly significant correlation between the rank order of potency for inhibition of FPP synthase in vitro and inhibition of bone resorption in vivo (13). Although FPP synthase appears to be the major pharmacological target of N-BPs, we have examined whether these compounds may also inhibit other enzymes in the mevalonate pathway, such as isopentenyl diphosphate (IPP) isomerase, the enzyme proximal to FPP synthase in the mevalonate pathway (Fig. 1). In this study we describe a novel N-BP, NE21650, that inhibits both FPP synthase and IPP isomerase, and NE10571, a pharmacologically inactive isomer of NE21650. MATERIALS AND METHODS Reagents. The sodium salts of alendronate (ALN; 4-aminobutane-1,1-bisphosphonic acid), NE21650 (2-aminophenyl-2-ethane1-hydroxy-1,1-bisphosphonic acid) and NE10571 (4-aminophenyl-2ethane-1,1-bisphosphonic acid) were synthesized by Proctor & Gamble Pharmaceuticals (Cincinnati, OH). The N-BPs were dissolved in PBS, the pH adjusted to 7.4 with 1 N NaOH, and then filtersterilized using a 0.2-m filter. All other reagents were obtained from Sigma Chemical Co., unless otherwise stated. [ 14C]Mevalonic acid lactone was purchased from NEN (Hounslow, UK).
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Expression of recombinant human IPP isomerase and FPP synthase. The human IPP isomerase clone pFMH12 in the bacterial expression vector pARC306N (14) was kindly provided by C. D. Poulter, University of Utah and expressed as previously described (13). Human FPP synthase (Clone KIA1293, a kind gift from the Kazusa DNA Research Institute, Kisarazu, Chiba, Japan) was expressed and partially purified as described previously (13). In vitro assay of IPP isomerase and FPP synthase activity. IPP isomerase activity was assayed by the acid-lability method (15) using [ 14C]IPP as a substrate, as previously described (13). The hydrolyzed reaction products were solvent extracted into ligroin and the radioactivity in the ligroin fraction (dimethylallyl diphosphate, the product of the isomerase reaction) was determined using a Packard Tricarb 1900CA scintillation counter. N-BPs were pre-incubated with the enzyme for 10 min prior to initiation of the reaction. FPP synthase activity was assayed using [ 14C]IPP and geranyl diphosphate as substrates, as described previously (13). Reaction products were extracted with water-saturated butyl alcohol and the amount of radioactivity in the upper phase was determined by scintillation counting, using a Packard Tricarb 1900CA scintillation counter. To determine the effects of N-BPs on FPP synthase activity, N-BPs were pre-incubated with the enzyme preparation for 10 min, prior to initiation of the reaction. Effect of N-BPs on Rap1A prenylation in osteoclasts and macrophages. Osteoclasts were isolated from the long bones of 2-day-old New Zealand white rabbits, seeded into 6-well culture plates then purified according to the method of Coxon et al. (10). J774 cells were cultured in 12-well plates (1 ⫻ 10 5 cells/well). After treatment with 50 M N-BPs for 24 h, J774 cells and osteoclasts were lysed in 0.1 ml 1% (v/v) NP-40, 0.1% (w/v) sodium dodecyl sulfate, 0.5% (w/v) sodium deoxycholate, 1% (v/v) protease inhibitor cocktail (Sigma). Twenty micrograms of J774 cell lysate or 50 g of rabbit osteoclast lysate was then electrophoresed on 12% polyacrylamide–SDS gels under reducing conditions. Following electrophoresis, the proteins were transferred to polyvinyldifluoride (PVDF) membrane and then hybridized with 0.2 g/ml goat polyclonal anti-Rap1A antibody (Santa Cruz Biotechnology Inc.), which recognizes only the unprenylated form of Rap1A (16, 17) followed by 1 g/ml anti-goat IgG– horseradish peroxidase (HRP) conjugate (Sigma). Chemiluminescent bands were visualized using Supersignal reagent (Pierce) and a Bio-Rad Fluor-S Max MultiImager. Incorporation of [ 14C]mevalonate into prenylated proteins in macrophages. Protein prenylation in J774 macrophages was studied by metabolically labeling cells with [ 14C]mevalonic acid lactone for 20 h in the presence of 50 or 100 M N-BPs, as described previously (4). Cell lysates were then examined for the presence of radiolabeled, prenylated proteins. Fifty micrograms of protein from each cell lysate was separated on 12% polyacrylamide–SDS gels under reducing conditions, then prenylated proteins were visualized by phosphorimaging, using a Kodak phosphorscreen and Bio-Rad Personal FX imager. Effects of N-BPs on osteoclast-mediated bone resorption. Rabbit osteoclasts were seeded on to 5-mm-diameter elephant tusk ivory slices in 96-well plates and allowed to adhere for 2 h. Following 48 h exposure with 0.1–100 M N-BPs, the slices were fixed with 4% formaldehyde and resorptive activity of osteoclasts was assessed by reflected light microscopy using a Leitz Quantimet Q500MC image analysis system, as described by van’t Hof et al. (18).
RESULTS AND DISCUSSION In this study we screened N-BPs for the ability to inhibit FPP synthase and IPP isomerase. NE21650, a bisphosphonate containing an amino group in the ortho-position of an aromatic ring (Fig. 1), was a potent
FIG. 1. Schematic representation of the mevalonate pathway, and the structures of nitrogen-containing bisphosphonates.
inhibitor of recombinant human FPP synthase, comparable in potency to ALN (IC 50 58 and 53 nM, respectively; Fig. 2). However, unlike ALN (Fig. 2) and other N-BPs studied previously, such as ibandronate, risedronate and incadronate (13), NE21650 also inhibited recombinant human IPP isomerase in vitro (IC 50 ⬃70 M; Fig. 2). NE10571 (an isomer of NE21650 containing a para-amino group rather than an ortho-amino group), had no inhibitory effect on IPP isomerase at concentrations up to 300 M, and was almost 700-fold less potent than NE21650 at inhibiting recombinant human FPP synthase. This indicates that the position of the amino group in an aromatic ring is an important determinant for inhibition of IPP isomerase and FPP synthase by bisphosphonates. This is in accord with our recent observation that the three-dimensional position of a nitrogen within a heterocyclic ring is critical for effective inhibition of FPP synthase by bisphosphonates (13). We next investigated whether, by inhibiting both FPP synthase and IPP isomerase, NE21650 was a more effective inhibitor of protein prenylation in vitro than ALN (which inhibits FPP synthase only, with a very similar IC 50 to NE21650). Western blot analysis, using an antibody that selectively recognizes the unprenylated form of Rap1A (16, 17), showed that NE21650 was a more effective inhibitor of protein pre-
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FIG. 3. Inhibition of prenylation of Rap1A by ALN and NE21650, but not NE10571, in J774 macrophages (A) and purified rabbit osteoclasts (B). Cells were treated with 50 M N-BPs for 24 h prior to separation of proteins in cell lysates by SDS–PAGE, then Western blotting using an antibody that recognizes the 21-kDa, unprenylated form of Rap1A (arrowheads). Blots were also probed with anti-actin antibody to ensure equal loading per lane (not shown). Data are representative of three independent experiments.
FIG. 2. (A) Inhibition of recombinant human IPP isomerase by NE21650, but not NE10571 or ALN. N-BPs were incubated with recombinant human IPP isomerase for 10 min prior to assaying activity. [ 14C]DMAPP formed in the reaction was extracted into ligroin and the activity determined by liquid scintillation counting. Data are the mean ⫾ SEM of at least three independent experiments, expressed as a percentage of IPP isomerase activity in the absence of N-BP. (B) Inhibition of recombinant human FPP synthase by NE21650 and ALN. N-BPs were incubated with recombinant human FPP synthase for 10 min prior to assaying for FPP synthase activity. 14C-labeled FPP and geranyl diphosphate formed in the reaction were extracted into water-saturated butanol and activity determined by liquid scintillation counting. Data are means ⫾ SEM of at least three independent experiments, expressed as a percentage of FPP synthase activity in the absence of N-BP.
nylation than ALN in both J774 macrophages and purified rabbit osteoclasts, since unprenylated Rap1A accumulated to a greater extent in cells treated with NE21650 compared to cells treated with ALN (Fig. 3). This suggests that inhibition of both IPP isomerase and FPP synthase prevents protein prenylation more effectively in intact cells than inhibition of FPP synthase alone. NE10571 did not cause accumulation of unprenylated Rap1A in J774 cells (Fig. 3), consistent with the lack of effect of this compound on IPP isomerase or FPP synthase. The greater effectiveness of NE21650 for inhibiting protein prenylation was confirmed by labeling J774 macrophages with [ 14C]mevalonate. At a concentration of 50 M, NE21650 clearly prevented the synthesis of isoprenoid lipids, that migrate at the dye front of SDS– PAGE gels (4, 10), but only slightly prevented the
prenylation of 21- to 26-kDa small GTPases (Fig. 4). ALN (50 M) had little effect on the synthesis of isoprenoid lipids or on protein prenylation. At a concentration of 100 M, NE21650 completely prevented the synthesis of isoprenoid lipids and very markedly prevented prenylation of small GTPases, whereas ALN was less effective. As expected, NE10571 had no effect on the synthesis of isoprenoid lipids or on protein prenylation (Fig. 4). Finally, given the apparent increase in the effectiveness of NE21650 for inhibiting protein prenylation, we examined whether this would confer an increase in antiresorptive potency compared to ALN. In an in vitro bone resorption assay using rabbit osteoclasts, NE21650 was slightly (but significantly) more effective than ALN at inhibiting resorption at concentrations ⱖ10 M (Fig. 5). Hence, NE21650, by inhibiting both
FIG. 4. Inhibition of protein prenylation by ALN and NE21650, but not NE10571, in J774 macrophages. Cells were metabolically labeled for 20 h with [ 14C]mevalonate in the absence or presence of 50 or 100 M ALN, NE10571, or NE21650. Cell lysates were analyzed by SDS–PAGE on 12% polyacrylamide gels and radiolabeled, prenylated proteins were detected by phosphorimaging. The position of isoprenoid intermediates and 21- to 26-kDa, prenylated small GTPases are indicated.
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ACKNOWLEDGMENTS We are grateful to C. D. Poulter and F. M. Hahn for providing the cDNA clone for IPP isomerase.
REFERENCES
FIG. 5. Inhibition of osteoclastic bone resorption by N-BPs in rabbit osteoclasts. Cells were treated with 0.1–100 M N-BPs for 48 h prior to quantification of the area of resorbed mineral by reflected light microscopy. Data are means ⫾ SEM of at least three independent experiments, expressed as a percentage of the area of mineral resorbed in untreated cultures.
IPP isomerase and FPP synthase, is more effective than ALN at inhibiting protein prenylation in osteoclasts and, consequently, has slightly increased antiresorptive potency. NE10571 did not inhibit bone resorption at all concentrations studied (up to 100 M). This study emphasizes the critical importance of the three-dimensional position of the nitrogen group in the bisphosphonate side chain (for example, ortho- vs paraposition in an aromatic ring) for determining the ability to inhibit FPP synthase and hence for determining the potency for inhibiting bone resorption. Furthermore, our findings illustrate that certain side-chain structures, such as NE21650, also allow the inhibition of other enzymes in the mevalonate pathway, such as IPP isomerase. The N-BPs ibandronate and incadronate, as well as inhibiting FPP synthase, are also known to inhibit squalene synthase (19, 20), thereby preventing cholesterol biosynthesis (Fig. 1). However, inhibition of squalene synthase does not prevent protein prenylation, and replenishing osteoclasts with cholesterol does not overcome the effects of N-BPs (5). Furthermore, the antiresorptive potency of ibandronate and incadronate correlates well with their ability to inhibit FPP synthase alone (13). Hence, inhibition of squalene synthase does not appear to directly enhance the antiresorptive effect of ibandronate or incadronate. By contrast, inhibition of IPP isomerase synthase by NE21650 appears to enhance the ability to prevent protein prenylation in intact cells and hence leads to a slight increase in antiresorptive potency compared to bisphosphonates that prevent protein prenylation by inhibiting only FPP synthase. The exact threedimensional structure of NE21650 that allows inhibition of IPP isomerase and FPP synthase remains to be determined.
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