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Recent advances in understanding the mechanism of action of bisphosphonates
Recent advances in understanding the mechanism of action of bisphosphonates Fraser P Coxon, Keith Thompson and Michael J Rogers Bisphosphonates (BPs) are widely used in the treatment of diseases associated with excessive osteoclast-mediated bone resorption, such as osteoporosis. Although several years ago the molecular target of the potent nitrogen-containing BPs (N-BPs) was identified as farnesyl diphosphate synthase, an enzyme in the mevalonate pathway, recent data have shed new light on the precise mechanism of inhibition and demonstrated that the acute-phase reaction, an adverse effect of N-BPs, is also caused by inhibition of this enzyme. In addition, the identification of BP analogues that inhibit different enzymes in the mevalonate pathway could lead to the development of novel inhibitors of bone resorption with potential applications in the treatment of bone disease. Addresses Bone Research Group, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK Corresponding author: Coxon, Fraser P ([email protected])
Current Opinion in Pharmacology 2006, 6:307–312 This review comes from a themed issue on Musculoskeletal Edited by Geoff Goldspink and Brendon Noble
1471-4892/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.coph.2006.03.005
Introduction Bisphosphonates (BPs) are the most widely used and effective antiresorptive agent for the treatment of diseases in which there is an increase in osteoclastic resorption, including post-menopausal osteoporosis, Paget’s disease and tumour-associated osteolysis. These drugs are synthetic analogues of inorganic pyrophosphate (PPi), consisting of two phosphonate groups linked by nonhydrolysable phosphoether bonds to a central carbon atom, to which are attached two covalently bonded sidechains (R1 and R2). This P–C–P backbone structure enables BPs to bind avidly to divalent metal ions such as Ca2+ [1], and as a result BPs bind to bone mineral surfaces in vivo [2]. This bone-targeting property, followed by their localized release during osteoclast-mediated resorption, explains why BPs appear to have a highly selective effect on osteoclasts in vivo [1]. BPs can be separated into two groups according to their molecular mechanism of action. The simple BPs that www.sciencedirect.com
most closely resemble PPi, such as clodronate and etidronate, are metabolized intracellularly into methylenecontaining analogues of ATP by aminoacyl-tRNA synthetase enzymes, with the nonhydrolysable P–C–P group of the BP replacing P–O–P (i.e. the b,g-phosphate groups) in ATP [3]. These AppCp-type metabolites accumulate in the cytosol of osteoclasts and induce cell death [4], probably by inhibiting ATP-utilizing enzymes such as adenine nucleotide translocase (ANT), a component of the mitochondrial permeability transition pore [5]. Inhibition of ANT by the AppCCl2p metabolite of clodronate causes hyperpolarization of the mitochondrial inner membrane [5], which can lead to breakdown of the mitochondrial membrane potential, release of cytochrome C and induction of apoptosis [1]. The more potent N-BPs, which have bulkier side-chains characterized by a nitrogen moiety either in an alkyl chain (e.g. alendronate and ibandronate) or within a heterocyclic structure (e.g. risedronate and zoledronate), are not metabolized. Rather, N-BPs act by inhibiting farnesyl diphosphate synthase (FPP synthase), an enzyme of the mevalonate pathway, thereby depleting cells of the isoprenoid lipids FPP and geranylgeranyl diphosphate (GGPP), which are required for the post-translational prenylation of small GTPases, such as those of the Ras, Rho and Rab families [6–8]. Because prenylation is required for the localization of these GTPases to subcellular membranes, N-BPs are thought to inhibit resorption by disrupting the localization and function of small GTPases that are essential for osteoclast activity and survival [1]. This review focuses on recent advances that have further clarified the mechanism of action of N-BPs, from the initial binding to bone and uptake into osteoclasts through to the mechanism of inhibition of FPP synthase and the consequences of inhibiting this enzyme.
Bone targeting of bisphosphonates and uptake by osteoclasts Although the bone-targeting property of BPs is known to be related to their ability to chelate Ca2+ ions through bidentate or tridentate binding via the phosphonate groups and R1 sidechain [1], recent studies suggest that the R2 sidechain of N-BPs can also influence overall bone affinity as a result of the ability of the nitrogen moiety to interact with the crystal surface of bone mineral. For example, risedronate and ibandronate appear to have a lower affinity for bone mineral than does alendronate or zoledronate [9]. Although the Current Opinion in Pharmacology 2006, 6:307–312
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detection and quantification of differences in bone affinity between BPs is controversial [10], it has been suggested that such differences in bone mineral affinity could account for the apparent differences in onset/ offset effects and duration of action of BPs on bone turnover. Because the ability of BPs to chelate Ca2+ is reduced at acidic pH, osteoclasts are exposed to a locally high concentration of BPs during resorption [11]. Recent studies with fluorescently labelled BPs have shown that osteoclasts then internalize BPs into membrane-bound vesicles by fluid-phase endocytosis [12] (F. Coxon et al., abstract in Bone 2006, 34:S51). BPs are probably endocytosed as microprecipitates with Ca2+, as chelation of Ca2+ with EGTA or the non-N-BP clodronate prevents the uptake and pharmacological effects of alendronate [12,13]. In addition to enabling release from the mineral surface, the activity of H+-ATPases is also essential for the uptake of BPs into the cytosol of osteoclasts [12,14], probably because acidification of endocytic vesicles reduces the negative charge on the phosphonate groups of BPs, favouring dissociation from Ca2+ ions and allowing either diffusion or transport of BPs across the vesicular membrane [12]. It remains unclear whether cell types other than osteoclasts in the bone microenvironment can internalize BPs from the bone surface, but recent data demonstrates that N-BPs have no detectable effect on protein prenylation in non-osteoclast bone cells when administered in vivo at doses that cause a robust inhibition of protein prenylation in osteoclasts [15,16]. In accordance with these data, in vitro studies have demonstrated that mineral-bound BPs have little effect on the proliferation or activity of osteoblasts or macrophages [17] (F. Coxon et al., abstract in Bone 2006, 38:S45).
Mechanism of inhibition of FPP synthase by N-BPs The exact mechanism by which N-BPs inhibit FPP synthase is only just becoming clear. The recent generation of X-ray crystal structures of human FPP synthase cocrystallized with N-BPs [18,19] indicates that the BPs appear to bind in one of the two isoprenoid lipid-binding pockets (the GPP/DMAPP pocket) in the enzyme active site, with the R2 sidechain positioned in the hydrophobic cleft that normally accommodates the isoprenoid lipid, and the phosphonate groups bonding to lysine residues and a cluster of three magnesium ions. Interestingly, the nitrogen atom in the R2 sidechain appears to form hydrogen bonds with a crucial, conserved threonine residue, consistent with the earlier proposal that N-BPs mimic the enzyme’s natural substrates GPP/DMAPP and act competitively as carbocation transition state analogues [20]. N-BPs also appear to inhibit bacterial FPP synthase in a similar manner [21]. Current Opinion in Pharmacology 2006, 6:307–312
Enzyme kinetic analysis with human FPP synthase indicates that the interaction with N-BPs is highly complex and characteristic of ‘slow-tight binding’ inhibition [19]. Initially, N-BPs appear to compete directly for binding with DMAPP or GPP. This is followed by more complex conformational changes that promote the binding of the second substrate, IPP, which itself stabilizes the final ternary complex of N-BP-bound enzyme, helping to explain the extraordinary inhibitory potency of some N-BPs towards this enzyme. Recent computer modelling studies suggested that a second molecule of N-BP might also bind to the IPP pocket in the enzyme, which appeared to be supported by enzyme kinetic analysis showing biphasic modes of inhibition [22]. However, this was not confirmed in X-ray crystal structures [18,19], and the kinetic studies might reflect more complex conformational changes in the structure of the enzyme as a result of the binding of N-BP in the DMAPP/GPP pocket. Hence, these studies are only beginning to reveal in detail why minor changes to the structure and conformation of N-BPs markedly influence antiresorptive potency [1]. The exceptional potency of N-BPs for inhibition of FPP synthase is important because it is likely that only small amounts of internalized N-BPs enter the cytosol to become available for inhibition of FPP synthase [12]. This is supported by the recent demonstration that alkylamines (which are likely to be much more accessible to cells as they resemble the R2 sidechain of N-BPs but lack the phosphonate groups) are 106 times weaker inhibitors of FPP synthase than are BPs, but only 103 times less potent inhibitors of protein prenylation [23]. Given the similarity between the substrates used, it is likely that N-BPs are also able to inhibit other enzymes of the mevalonate pathway. Indeed, some N-BPs can inhibit GGPP synthase [24], although the much lower potency towards this enzyme suggests that this effect might not contribute to the overall pharmacological effects of NBPs on osteoclasts.
Consequences of inhibiting FPP synthase Small GTPases, such as those of the Ras, Rho and Rab families, are crucial signalling proteins that regulate a variety of cell processes necessary for osteoclast function, including cytoskeletal arrangement, membrane ruffling, trafficking of intracellular vesicles and cell survival [25]. It has been assumed that N-BPs, by preventing protein prenylation, disrupt the interaction of these small GTPases with cell membranes, thereby interfering with these processes. For example, loss of prenylation of Rho, Rac or Cdc42 is likely to disrupt F-actin rings, a specialized cytoskeletal structure unique to osteoclasts, whereas inhibition of prenylation of Rab proteins disrupts the ruffled border [26], a convoluted region of plasma membrane that is essential for resorption, probably by disrupting the trafficking of intracellular vesicles required to form this membrane domain. www.sciencedirect.com
Recent advances in understanding the mechanism of action of bisphosphonates Coxon, Thompson and Rogers 309
Surprisingly, we have recently demonstrated that the unprenylated forms of Rho family GTPases that accumulate after treatment with N-BPs are in the active GTPbound form, most likely because of their inability to interact with regulatory proteins such as RhoGAP [27]. Moreover, although these unprenylated proteins are mislocalized and would therefore not be expected to be able to activate downstream signalling pathways, N-BPs cause sustained activation of p38 downstream of Rac, at least in macrophages [27]. There is also limited evidence that unprenylated Rho-family proteins can interact productively with a physiological effector in other systems [28]. This raises the intriguing possibility that N-BPs affect osteoclasts by inappropriate stimulation, rather than inhibition, of small GTPase-mediated signalling pathways. In support of this, other studies have shown that inhibition of protein prenylation with a farnesyl transferase inhibitor or statins cause increased levels of unprenylated, GTPbound Ras and Rac, respectively, although it remains unclear whether the unprenylated proteins activated downstream signalling [29–31]. Therefore, rather than acting by causing the loss of pre-existing, prenylated small GTPases and hence loss of signalling through these proteins, the ability of BPs to inhibit osteoclast function might be caused, at least in part, by the accumulation of the unprenylated form of small GTPases, resulting in inappropriate activation of downstream signalling pathways, and/or a dominant negative effect on small GTPase signalling due to the sequestration of effector proteins in nonproductive cytoplasmic complexes with unprenylated GTPases (which has previously been described for Ras signalling [32]) (Figure 1). Further studies are required to
determine which mechanisms occur in osteoclasts and in other cell types that might be directly affected by N-BPs in vivo, such as tumour cells [33]. A recent report suggests another intriguing mechanism by which N-BPs could disrupt osteoclast function as a result of inhibition of FPP synthase. Inhibition of this enzyme causes the accumulation of the upstream substrate IPP, which then becomes conjugated to AMP to form a novel ATP analogue (ApppI). This metabolite, as with the AppCp-type metabolites of simple BPs, can inhibit mitochondrial ANT and induce osteoclast apoptosis [34]. However, the pharmacological significance of this is unclear, because restoring prenylation with a substrate for protein geranylgeranylation overcomes the antiresorptive effects of N-BPs in vitro [35], but would be unlikely to affect levels of ApppI. Furthermore, unlike the simple BPs that act by inducing osteoclast apoptosis, the antiresorptive effect of N-BPs is not dependent on apoptosis, at least in vitro [36]. Hence, inhibition of protein prenylation remains the most likely explanation for the antiresorptive effects of N-BPs. Interestingly however, recent studies by van Beek et al. [37] suggest that pamidronate might have an additional, as yet unidentified, molecular target in osteoclasts, as (unlike other N-BPs) its antiresorptive effect could not be overcome by replenishing cells with a substrate for protein prenylation.
Molecular basis for the acute-phase reaction to bisphosphonates The major adverse effect of intravenous N-BP administration is a flu-like acute-phase reaction, which typically occurs
Figure 1
Potential mechanisms by which N-BPs could affect signalling, following inhibition of prenylation of Rho family GTPases. (a) By inhibiting FPP synthase, N-BPs deplete the levels of prenylated proteins, perhaps leading to loss of downstream signalling pathways required for osteoclast function. Alternatively, accumulation of GTP-bound unprenylated GTPases might either (b) cause inappropriate activation of downstream signalling pathways or (c) exert a dominant negative effect on signalling by sequestering effectors in nonproductive cytoplasmic complexes. These potential effects could also apply to prenylated GTPases other than those of the Rho family, although this remains to be studied. www.sciencedirect.com
Current Opinion in Pharmacology 2006, 6:307–312
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in about one-third of patients receiving N-BPs for the first time. Although this phenomenon was first described 20 years ago [38], the molecular mechanism involved has only recently become clear. A study by Kunzmann et al. [39] reported that patients who suffered an acute-phase reaction to pamidronate had increased circulating levels of gd Tcells in peripheral blood. Unlike conventional ab T-cells, gd T-cells recognize low molecular weight non-peptide molecules without conventional major histocompatibility complex class presentation. The major subset of gd T-cells found in humans (Vg9Vd2+ — also termed Vg2Vd2+) are activated by a diverse array of these molecules, including pyrophosphomonoesters such as IPP, alkylamines and NBPs. It is now clear that inhibition of FPP synthase is the route by which N-BPs activate Vg9Vd2+ T cells, rather than direct binding of N-BPs to the Vg9Vd2 T cell receptor [40]. Following an intravenous infusion of N-BP, the plasma concentration (approximately 10 6 M for zoledronic acid) probably enables sufficient internalization of N-BP by highly endocytic cells such as monocytes to cause inhibition of FPP synthase and the subsequent intracellular accumulation of upstream metabolites, including IPP [40]. This intracellular IPP is somehow ‘presented’ to Vg9Vd2+ T cells in the peripheral circulation, causing their activation and proliferation and the release of the pro-inflammatory cytokines TNFa and IFNg, which are characteristic of the acute-phase reaction (Figure 2). In support of this idea, Vg9Vd2 T cells have been shown to recognize endogenous mevalonate metabolites, such as IPP produced by zoledronate-pulsed tumour cells [41],
dependent upon internalization of the N-BP. Independently, it was also demonstrated that the ability of N-BPs to activate Vg9Vd2+ T cells closely matches the ability to inhibit FPP synthase [40,42], and that co-treatment of cells with a statin (to prevent the accumulation of IPP) completely abrogates the stimulatory potential of N-BPs on gd T cell activation and proliferation [40]. The latter observation raises the intriguing possibility that statins could be used to prevent the acute-phase reaction to NBPs. The acute-phase reaction to N-BPs differs from a typical acute-phase response in that N-BP-treated Vg9Vd2 T cells, rather than CD14+ monocytes and macrophages, are the primary source of the proinflammatory cytokines tumour necrosis factor-a and interleukin-6 [43]. Thus, CD14+ cells are necessary for IPP production and presentation to Vg9Vd2+ T-cells, whereas Vg9Vd2+ T cells are responsible for the cytokine release characteristic of the acute-phase reaction to N-BPs in vivo (Figure 2).
Potential of new bisphosphonate analogues Recently, it has become clear that changes to the structure of N-BPs can give rise to compounds capable of inhibiting other enzymes of the mevalonate pathway that use isoprenoid lipids. For example, we found that a weakly antiresorptive phosphonocarboxylate (PC) analogue of risedronate, in which one of the phosphonate groups of risedronate is replaced with a carboxylate group (3-PEHPC, previously called NE10790), has no effect on FPP synthase but specifically inhibits the protein prenyl
Figure 2
Proposed mechanism of the acute-phase reaction to N-BPs. Following infusion, N-BPs are internalized by endocytic cells (probably monocytes) in the peripheral circulation and inhibit FPP synthase, causing accumulation of the upstream metabolite IPP. Recognition of IPP by Vg9Vd2+ T cells then leads to activation of gd T cells and release of the proinflammatory cytokines that cause the symptoms of the acute phase reaction. Current Opinion in Pharmacology 2006, 6:307–312
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Recent advances in understanding the mechanism of action of bisphosphonates Coxon, Thompson and Rogers 311
transferase Rab GGTase (RGGT) and, therefore, inhibits the prenylation and membrane localization of only the Rab GTPases [16,26]. Other PCs are also able to inhibit RGGT, but the structure–activity relationships do not match the structure–activity relationships of the parent BPs for inhibiting FPP synthase [16], indicating that the sidechains of N-BPs and PCs interact differently with their respective target enzymes, despite the similarity in isoprenoid substrates of FPP synthase and RGGT. Interestingly, unlike N-BPs, 3-PEHPC does not activate gd T cells in vitro [40] because of its lack of effect on FPP synthase, and is therefore unlikely to cause an acutephase reaction in vivo. A further potential advantage of such compounds is their relatively low bone affinity, as they lack one of the phosphonate groups of BPs. This might be an attractive property in situations where longterm retention in bone is undesirable, such as the treatment of paediatric bone disease. In addition, compounds with low bone affinity might have greater potential than BPs for direct effects on cells within bone other than osteoclasts, such as tumour cells. Because 3-PEHPC, like its parent N-BP risedronate, can inhibit invasion of breast cancer cells and induce apoptosis of myeloma cells in vitro [44,45], low affinity RGGT inhibitors such as PCs are potential novel treatments for metastatic bone disease. This possibility is strengthened by the intriguing finding that some drugs that were designed to inhibit farnesyl transferase, and have shown anticancer activity, are also able to inhibit RGGT and are likely to act through disruption of Rab prenylation, at least in some tumour cell types [46].
Conclusions
2.
Masarachia P, Weinreb M, Balena R, Rodan GA: Comparison of the distribution of 3H-alendronate and 3H-etidronate in rat and mouse bones. Bone 1996, 19:281-290.
3.
Frith JC, Monkkonen J, Blackburn GM, Russell RG, Rogers MJ: Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5(-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro. J Bone Miner Res 1997, 12:1358-1367.
4.
Frith JC, Monkkonen J, Auriola S, Monkkonen H, Rogers MJ: The molecular mechanism of action of the anti-resorptive and anti-inflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis. Arthritis Rheum 2001, 44:2201-2210.
5.
Lehenkari PP, Kellinsalmi M, Napankangas JP, Ylitalo KV, Monkkonen J, Rogers MJ, Azhayev A, Vaananen HK, Hassinen IE: Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite. Mol Pharmacol 2002, 61:1255-1262.
6.
van Beek E, Pieterman E, Cohen L, Lowik C, Papapoulos S: Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates. Biochem Biophys Res Commun 1999, 264:108-111.
7.
Bergstrom JD, Bostedor RG, Masarachia PJ, Reszka AA, Rodan G: Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch Biochem Biophys 2000, 373:231-241.
8.
Dunford JE, Thompson K, Coxon FP, Luckman SP, Hahn FM, Poulter CD, Ebetino FH, Rogers MJ: Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogencontaining bisphosphonates. J Pharmacol Exp Ther 2001, 296:235-242.
9.
Nancollas GH, Tang R, Phipps RJ, Henneman Z, Gulde S, Wu W, Mangood A, Russell RG, Ebetino FH: Novel insights into actions of bisphosphonates on bone: Differences in interactions with hydroxyapatite. Bone 2006, in press.
10. Leu CT, Luegmayr E, Freedman LP, Rodan GA, Reszka AA: Relative binding affinities of bisphosphonates for human bone and relationship to antiresorptive efficacy. Bone 2006, in press.
FPP synthase is the major pharmacological target of NBPs, accounting for not only the antiresorptive activity of these drugs, but also their ability to induce an acute-phase reaction, via the activation of gd T cells. Recent studies have clarified the exact mechanism by which N-BPs inhibit this enzyme, thus finally explaining the relationship between chemical structure and antiresorptive potency of these blockbuster drugs. However, further studies are needed to understand the subsequent cellular events in osteoclasts, as it remains unclear whether it is the loss of prenylated small GTPases or the parallel accumulation of unprenylated, mislocalized small GTPases that accounts for the inhibitory effects of NBPs on osteoclast function and survival.
11. Ebetino FH, Francis MD, Rogers MJ, Russell RGG: Mechanisms of action of etidronate and other bisphosphonates. Rev Contemp Pharmacother 1998, 9:233-243.
References and recommended reading
16. Coxon FP, Ebetino FH, Mules EH, Seabra MC, McKenna CE, Rogers MJ: Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the prenylation and membrane localization of Rab proteins in osteoclasts in vitro and in vivo. Bone 2005, 37:349-358.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Rogers MJ: New insights into the molecular mechanisms of action of bisphosphonates. Curr Pharm Des 2003, 9:2643-2658.
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12. Thompson K, Rogers MJ, Coxon FP, Crockett JC: Cytosolic entry of bisphosphonate drugs requires acidification of vesicles following fluid-phase endocytosis. Mol Pharmacol 2006, in press. 13. Frith JC, Rogers MJ: Antagonistic effects of different classes of bisphosphonates in osteoclasts and macrophages in vitro. J Bone Miner Res 2003, 18:204-212. 14. Takami M, Suda K, Sahara T, Itoh K, Nagai K, Sasaki T, Udagawa N, Takahashi N: Involvement of vacuolar H(+)-ATPase in incorporation of risedronate into osteoclasts. Bone 2003, 32:341-349. 15. Staal A, Frith JC, French MH, Swartz J, Gungor T, Harrity TW, Tamasi J, Rogers MJ, Feyen JH: The ability of statins to inhibit bone resorption is directly related to their inhibitory effect on HMG-CoA reductase activity. J Bone Miner Res 2003, 18:88-96.
17. Schindeler A, Little DG: Osteoclasts but not osteoblasts are affected by a calcified surface treated with zoledronic acid in vitro. Biochem Biophys Res Commun 2005, 338:710-716. 18. Rondeau JM, Bitsch F, Geiser M, Hemmig R, Kroemer M, Lehmann S, Ramage P, Rieffel S, Strauss A, Green JR, Jahnke W: Current Opinion in Pharmacology 2006, 6:307–312
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Structural basis for the exceptional in vivo efficacy of bisphosphonate drugs. ChemMedChem 2006, 1:267-273. See annotation to [19]. 19. Kavanagh K, Guo K, Dunford JE, Wu X, Knapp S, Ebetino FH, Rogers MJ, Russell RGG, Oppermann U: The molecular mechanism of nitrogen-containing bisphosphonates as anti-osteoporosis drugs: crystal structure and inhibition of farnesyl pyrophosphate synthase. Proc Natl Acad Sci U S A 2006, in press. The authors of this and the previous article reported, independently of each other, the first X-ray crystal structure of human FPP synthase and identified the mode of inhibition of the enzyme by N-BPs and residues crucial for drug–protein interactions. 20. Martin MB, Arnold W, Heath HT, Urbina JA, Oldfield E: Nitrogen-containing bisphosphonates as carbocation transition state analogs for isoprenoid biosynthesis. Biochem Biophys Res Commun 1999, 263:754-758. 21. Hosfield DJ, Zhang Y, Dougan DR, Brooun A, Tari LW, Swanson RV, Finn J: Structural basis for bisphosphonatemediated inhibition of isoprenoid biosynthesis. J Biol Chem 2003, 279:8526-8529. 22. Ebetino FH, Roze CN, McKenna CE, Barnett BL, Dunford JE, Russell RGG, Mieling GE, Rogers MJ: Molecular interactions of nitrogen-containing bisphosphonates within farnesyl diphosphate synthase. J Organomet Chem 2005, 690:2679-2687. 23. Thompson K, Rojas-Navea J, Rogers MJ: Alkylamines cause Vg9Vd2 T-cell activation and proliferation by inhibiting the mevalonate pathway. Blood 2006, 107:651-654. 24. Szabo CM, Matsumura Y, Fukura S, Martin MB, Sanders JM, Sengupta S, Cieslak JA, Loftus TC, Lea CR, Lee HJ et al.: Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates and diphosphates: a potential route to new bone antiresorption and antiparasitic agents. J Med Chem 2002, 45:2185-2196.
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43. Hewitt RE, Lissina A, Green AE, Slay ES, Price DA, Sewell AK: The bisphosphonate acute phase response: rapid and copious production of proinflammatory cytokines by peripheral blood gd T cells in response to aminobisphosphonates is inhibited by statins. Clin Exp Immunol 2005, 139:101-111. 44. Boissier S, Ferreras M, Peyruchaud O, Magnetto S, Ebetino FH, Colombel M, Delmas P, Delaisse JM, Clezardin P: Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases. Cancer Res 2000, 60:2949-2954. 45. Roelofs AJ, Hulley PA, Meijer A, Ebetino FH, Russell RGG, Shipman CM: Selective inhibition of Rab prenylation by a phosphonocarboxylate analogue of risedronate induces apoptosis, but not S-phase arrest, in human myeloma cells. Int J Cancer 2006, in press. 46. Lackner MR, Kindt RM, Carroll PM, Brown K, Cancilla MR, Chen C, de Silva H, Franke Y, Guan B, Heuer T et al.: Chemical genetics identifies Rab geranylgeranyl transferase as an apoptotic target of farnesyl transferase inhibitors. Cancer Cell 2005, 7:325-336.
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Current Opinion in Pharmacology 2006, 6:307–312 This review comes from a themed issue on Musculoskeletal Edited by Geoff Goldspink and Brendon Noble
1471-4892/$ – see front matter Published by Elsevier Ltd. DOI 10.1016/j.coph.2006.03.005
Introduction Bisphosphonates (BPs) are the most widely used and effective antiresorptive agent for the treatment of diseases in which there is an increase in osteoclastic resorption, including post-menopausal osteoporosis, Paget’s disease and tumour-associated osteolysis. These drugs are synthetic analogues of inorganic pyrophosphate (PPi), consisting of two phosphonate groups linked by nonhydrolysable phosphoether bonds to a central carbon atom, to which are attached two covalently bonded sidechains (R1 and R2). This P–C–P backbone structure enables BPs to bind avidly to divalent metal ions such as Ca2+ [1], and as a result BPs bind to bone mineral surfaces in vivo [2]. This bone-targeting property, followed by their localized release during osteoclast-mediated resorption, explains why BPs appear to have a highly selective effect on osteoclasts in vivo [1]. BPs can be separated into two groups according to their molecular mechanism of action. The simple BPs that www.sciencedirect.com
most closely resemble PPi, such as clodronate and etidronate, are metabolized intracellularly into methylenecontaining analogues of ATP by aminoacyl-tRNA synthetase enzymes, with the nonhydrolysable P–C–P group of the BP replacing P–O–P (i.e. the b,g-phosphate groups) in ATP [3]. These AppCp-type metabolites accumulate in the cytosol of osteoclasts and induce cell death [4], probably by inhibiting ATP-utilizing enzymes such as adenine nucleotide translocase (ANT), a component of the mitochondrial permeability transition pore [5]. Inhibition of ANT by the AppCCl2p metabolite of clodronate causes hyperpolarization of the mitochondrial inner membrane [5], which can lead to breakdown of the mitochondrial membrane potential, release of cytochrome C and induction of apoptosis [1]. The more potent N-BPs, which have bulkier side-chains characterized by a nitrogen moiety either in an alkyl chain (e.g. alendronate and ibandronate) or within a heterocyclic structure (e.g. risedronate and zoledronate), are not metabolized. Rather, N-BPs act by inhibiting farnesyl diphosphate synthase (FPP synthase), an enzyme of the mevalonate pathway, thereby depleting cells of the isoprenoid lipids FPP and geranylgeranyl diphosphate (GGPP), which are required for the post-translational prenylation of small GTPases, such as those of the Ras, Rho and Rab families [6–8]. Because prenylation is required for the localization of these GTPases to subcellular membranes, N-BPs are thought to inhibit resorption by disrupting the localization and function of small GTPases that are essential for osteoclast activity and survival [1]. This review focuses on recent advances that have further clarified the mechanism of action of N-BPs, from the initial binding to bone and uptake into osteoclasts through to the mechanism of inhibition of FPP synthase and the consequences of inhibiting this enzyme.
Bone targeting of bisphosphonates and uptake by osteoclasts Although the bone-targeting property of BPs is known to be related to their ability to chelate Ca2+ ions through bidentate or tridentate binding via the phosphonate groups and R1 sidechain [1], recent studies suggest that the R2 sidechain of N-BPs can also influence overall bone affinity as a result of the ability of the nitrogen moiety to interact with the crystal surface of bone mineral. For example, risedronate and ibandronate appear to have a lower affinity for bone mineral than does alendronate or zoledronate [9]. Although the Current Opinion in Pharmacology 2006, 6:307–312
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detection and quantification of differences in bone affinity between BPs is controversial [10], it has been suggested that such differences in bone mineral affinity could account for the apparent differences in onset/ offset effects and duration of action of BPs on bone turnover. Because the ability of BPs to chelate Ca2+ is reduced at acidic pH, osteoclasts are exposed to a locally high concentration of BPs during resorption [11]. Recent studies with fluorescently labelled BPs have shown that osteoclasts then internalize BPs into membrane-bound vesicles by fluid-phase endocytosis [12] (F. Coxon et al., abstract in Bone 2006, 34:S51). BPs are probably endocytosed as microprecipitates with Ca2+, as chelation of Ca2+ with EGTA or the non-N-BP clodronate prevents the uptake and pharmacological effects of alendronate [12,13]. In addition to enabling release from the mineral surface, the activity of H+-ATPases is also essential for the uptake of BPs into the cytosol of osteoclasts [12,14], probably because acidification of endocytic vesicles reduces the negative charge on the phosphonate groups of BPs, favouring dissociation from Ca2+ ions and allowing either diffusion or transport of BPs across the vesicular membrane [12]. It remains unclear whether cell types other than osteoclasts in the bone microenvironment can internalize BPs from the bone surface, but recent data demonstrates that N-BPs have no detectable effect on protein prenylation in non-osteoclast bone cells when administered in vivo at doses that cause a robust inhibition of protein prenylation in osteoclasts [15,16]. In accordance with these data, in vitro studies have demonstrated that mineral-bound BPs have little effect on the proliferation or activity of osteoblasts or macrophages [17] (F. Coxon et al., abstract in Bone 2006, 38:S45).
Mechanism of inhibition of FPP synthase by N-BPs The exact mechanism by which N-BPs inhibit FPP synthase is only just becoming clear. The recent generation of X-ray crystal structures of human FPP synthase cocrystallized with N-BPs [18,19] indicates that the BPs appear to bind in one of the two isoprenoid lipid-binding pockets (the GPP/DMAPP pocket) in the enzyme active site, with the R2 sidechain positioned in the hydrophobic cleft that normally accommodates the isoprenoid lipid, and the phosphonate groups bonding to lysine residues and a cluster of three magnesium ions. Interestingly, the nitrogen atom in the R2 sidechain appears to form hydrogen bonds with a crucial, conserved threonine residue, consistent with the earlier proposal that N-BPs mimic the enzyme’s natural substrates GPP/DMAPP and act competitively as carbocation transition state analogues [20]. N-BPs also appear to inhibit bacterial FPP synthase in a similar manner [21]. Current Opinion in Pharmacology 2006, 6:307–312
Enzyme kinetic analysis with human FPP synthase indicates that the interaction with N-BPs is highly complex and characteristic of ‘slow-tight binding’ inhibition [19]. Initially, N-BPs appear to compete directly for binding with DMAPP or GPP. This is followed by more complex conformational changes that promote the binding of the second substrate, IPP, which itself stabilizes the final ternary complex of N-BP-bound enzyme, helping to explain the extraordinary inhibitory potency of some N-BPs towards this enzyme. Recent computer modelling studies suggested that a second molecule of N-BP might also bind to the IPP pocket in the enzyme, which appeared to be supported by enzyme kinetic analysis showing biphasic modes of inhibition [22]. However, this was not confirmed in X-ray crystal structures [18,19], and the kinetic studies might reflect more complex conformational changes in the structure of the enzyme as a result of the binding of N-BP in the DMAPP/GPP pocket. Hence, these studies are only beginning to reveal in detail why minor changes to the structure and conformation of N-BPs markedly influence antiresorptive potency [1]. The exceptional potency of N-BPs for inhibition of FPP synthase is important because it is likely that only small amounts of internalized N-BPs enter the cytosol to become available for inhibition of FPP synthase [12]. This is supported by the recent demonstration that alkylamines (which are likely to be much more accessible to cells as they resemble the R2 sidechain of N-BPs but lack the phosphonate groups) are 106 times weaker inhibitors of FPP synthase than are BPs, but only 103 times less potent inhibitors of protein prenylation [23]. Given the similarity between the substrates used, it is likely that N-BPs are also able to inhibit other enzymes of the mevalonate pathway. Indeed, some N-BPs can inhibit GGPP synthase [24], although the much lower potency towards this enzyme suggests that this effect might not contribute to the overall pharmacological effects of NBPs on osteoclasts.
Consequences of inhibiting FPP synthase Small GTPases, such as those of the Ras, Rho and Rab families, are crucial signalling proteins that regulate a variety of cell processes necessary for osteoclast function, including cytoskeletal arrangement, membrane ruffling, trafficking of intracellular vesicles and cell survival [25]. It has been assumed that N-BPs, by preventing protein prenylation, disrupt the interaction of these small GTPases with cell membranes, thereby interfering with these processes. For example, loss of prenylation of Rho, Rac or Cdc42 is likely to disrupt F-actin rings, a specialized cytoskeletal structure unique to osteoclasts, whereas inhibition of prenylation of Rab proteins disrupts the ruffled border [26], a convoluted region of plasma membrane that is essential for resorption, probably by disrupting the trafficking of intracellular vesicles required to form this membrane domain. www.sciencedirect.com
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Surprisingly, we have recently demonstrated that the unprenylated forms of Rho family GTPases that accumulate after treatment with N-BPs are in the active GTPbound form, most likely because of their inability to interact with regulatory proteins such as RhoGAP [27]. Moreover, although these unprenylated proteins are mislocalized and would therefore not be expected to be able to activate downstream signalling pathways, N-BPs cause sustained activation of p38 downstream of Rac, at least in macrophages [27]. There is also limited evidence that unprenylated Rho-family proteins can interact productively with a physiological effector in other systems [28]. This raises the intriguing possibility that N-BPs affect osteoclasts by inappropriate stimulation, rather than inhibition, of small GTPase-mediated signalling pathways. In support of this, other studies have shown that inhibition of protein prenylation with a farnesyl transferase inhibitor or statins cause increased levels of unprenylated, GTPbound Ras and Rac, respectively, although it remains unclear whether the unprenylated proteins activated downstream signalling [29–31]. Therefore, rather than acting by causing the loss of pre-existing, prenylated small GTPases and hence loss of signalling through these proteins, the ability of BPs to inhibit osteoclast function might be caused, at least in part, by the accumulation of the unprenylated form of small GTPases, resulting in inappropriate activation of downstream signalling pathways, and/or a dominant negative effect on small GTPase signalling due to the sequestration of effector proteins in nonproductive cytoplasmic complexes with unprenylated GTPases (which has previously been described for Ras signalling [32]) (Figure 1). Further studies are required to
determine which mechanisms occur in osteoclasts and in other cell types that might be directly affected by N-BPs in vivo, such as tumour cells [33]. A recent report suggests another intriguing mechanism by which N-BPs could disrupt osteoclast function as a result of inhibition of FPP synthase. Inhibition of this enzyme causes the accumulation of the upstream substrate IPP, which then becomes conjugated to AMP to form a novel ATP analogue (ApppI). This metabolite, as with the AppCp-type metabolites of simple BPs, can inhibit mitochondrial ANT and induce osteoclast apoptosis [34]. However, the pharmacological significance of this is unclear, because restoring prenylation with a substrate for protein geranylgeranylation overcomes the antiresorptive effects of N-BPs in vitro [35], but would be unlikely to affect levels of ApppI. Furthermore, unlike the simple BPs that act by inducing osteoclast apoptosis, the antiresorptive effect of N-BPs is not dependent on apoptosis, at least in vitro [36]. Hence, inhibition of protein prenylation remains the most likely explanation for the antiresorptive effects of N-BPs. Interestingly however, recent studies by van Beek et al. [37] suggest that pamidronate might have an additional, as yet unidentified, molecular target in osteoclasts, as (unlike other N-BPs) its antiresorptive effect could not be overcome by replenishing cells with a substrate for protein prenylation.
Molecular basis for the acute-phase reaction to bisphosphonates The major adverse effect of intravenous N-BP administration is a flu-like acute-phase reaction, which typically occurs
Figure 1
Potential mechanisms by which N-BPs could affect signalling, following inhibition of prenylation of Rho family GTPases. (a) By inhibiting FPP synthase, N-BPs deplete the levels of prenylated proteins, perhaps leading to loss of downstream signalling pathways required for osteoclast function. Alternatively, accumulation of GTP-bound unprenylated GTPases might either (b) cause inappropriate activation of downstream signalling pathways or (c) exert a dominant negative effect on signalling by sequestering effectors in nonproductive cytoplasmic complexes. These potential effects could also apply to prenylated GTPases other than those of the Rho family, although this remains to be studied. www.sciencedirect.com
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in about one-third of patients receiving N-BPs for the first time. Although this phenomenon was first described 20 years ago [38], the molecular mechanism involved has only recently become clear. A study by Kunzmann et al. [39] reported that patients who suffered an acute-phase reaction to pamidronate had increased circulating levels of gd Tcells in peripheral blood. Unlike conventional ab T-cells, gd T-cells recognize low molecular weight non-peptide molecules without conventional major histocompatibility complex class presentation. The major subset of gd T-cells found in humans (Vg9Vd2+ — also termed Vg2Vd2+) are activated by a diverse array of these molecules, including pyrophosphomonoesters such as IPP, alkylamines and NBPs. It is now clear that inhibition of FPP synthase is the route by which N-BPs activate Vg9Vd2+ T cells, rather than direct binding of N-BPs to the Vg9Vd2 T cell receptor [40]. Following an intravenous infusion of N-BP, the plasma concentration (approximately 10 6 M for zoledronic acid) probably enables sufficient internalization of N-BP by highly endocytic cells such as monocytes to cause inhibition of FPP synthase and the subsequent intracellular accumulation of upstream metabolites, including IPP [40]. This intracellular IPP is somehow ‘presented’ to Vg9Vd2+ T cells in the peripheral circulation, causing their activation and proliferation and the release of the pro-inflammatory cytokines TNFa and IFNg, which are characteristic of the acute-phase reaction (Figure 2). In support of this idea, Vg9Vd2 T cells have been shown to recognize endogenous mevalonate metabolites, such as IPP produced by zoledronate-pulsed tumour cells [41],
dependent upon internalization of the N-BP. Independently, it was also demonstrated that the ability of N-BPs to activate Vg9Vd2+ T cells closely matches the ability to inhibit FPP synthase [40,42], and that co-treatment of cells with a statin (to prevent the accumulation of IPP) completely abrogates the stimulatory potential of N-BPs on gd T cell activation and proliferation [40]. The latter observation raises the intriguing possibility that statins could be used to prevent the acute-phase reaction to NBPs. The acute-phase reaction to N-BPs differs from a typical acute-phase response in that N-BP-treated Vg9Vd2 T cells, rather than CD14+ monocytes and macrophages, are the primary source of the proinflammatory cytokines tumour necrosis factor-a and interleukin-6 [43]. Thus, CD14+ cells are necessary for IPP production and presentation to Vg9Vd2+ T-cells, whereas Vg9Vd2+ T cells are responsible for the cytokine release characteristic of the acute-phase reaction to N-BPs in vivo (Figure 2).
Potential of new bisphosphonate analogues Recently, it has become clear that changes to the structure of N-BPs can give rise to compounds capable of inhibiting other enzymes of the mevalonate pathway that use isoprenoid lipids. For example, we found that a weakly antiresorptive phosphonocarboxylate (PC) analogue of risedronate, in which one of the phosphonate groups of risedronate is replaced with a carboxylate group (3-PEHPC, previously called NE10790), has no effect on FPP synthase but specifically inhibits the protein prenyl
Figure 2
Proposed mechanism of the acute-phase reaction to N-BPs. Following infusion, N-BPs are internalized by endocytic cells (probably monocytes) in the peripheral circulation and inhibit FPP synthase, causing accumulation of the upstream metabolite IPP. Recognition of IPP by Vg9Vd2+ T cells then leads to activation of gd T cells and release of the proinflammatory cytokines that cause the symptoms of the acute phase reaction. Current Opinion in Pharmacology 2006, 6:307–312
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Recent advances in understanding the mechanism of action of bisphosphonates Coxon, Thompson and Rogers 311
transferase Rab GGTase (RGGT) and, therefore, inhibits the prenylation and membrane localization of only the Rab GTPases [16,26]. Other PCs are also able to inhibit RGGT, but the structure–activity relationships do not match the structure–activity relationships of the parent BPs for inhibiting FPP synthase [16], indicating that the sidechains of N-BPs and PCs interact differently with their respective target enzymes, despite the similarity in isoprenoid substrates of FPP synthase and RGGT. Interestingly, unlike N-BPs, 3-PEHPC does not activate gd T cells in vitro [40] because of its lack of effect on FPP synthase, and is therefore unlikely to cause an acutephase reaction in vivo. A further potential advantage of such compounds is their relatively low bone affinity, as they lack one of the phosphonate groups of BPs. This might be an attractive property in situations where longterm retention in bone is undesirable, such as the treatment of paediatric bone disease. In addition, compounds with low bone affinity might have greater potential than BPs for direct effects on cells within bone other than osteoclasts, such as tumour cells. Because 3-PEHPC, like its parent N-BP risedronate, can inhibit invasion of breast cancer cells and induce apoptosis of myeloma cells in vitro [44,45], low affinity RGGT inhibitors such as PCs are potential novel treatments for metastatic bone disease. This possibility is strengthened by the intriguing finding that some drugs that were designed to inhibit farnesyl transferase, and have shown anticancer activity, are also able to inhibit RGGT and are likely to act through disruption of Rab prenylation, at least in some tumour cell types [46].
Conclusions
2.
Masarachia P, Weinreb M, Balena R, Rodan GA: Comparison of the distribution of 3H-alendronate and 3H-etidronate in rat and mouse bones. Bone 1996, 19:281-290.
3.
Frith JC, Monkkonen J, Blackburn GM, Russell RG, Rogers MJ: Clodronate and liposome-encapsulated clodronate are metabolized to a toxic ATP analog, adenosine 5(-(beta, gamma-dichloromethylene) triphosphate, by mammalian cells in vitro. J Bone Miner Res 1997, 12:1358-1367.
4.
Frith JC, Monkkonen J, Auriola S, Monkkonen H, Rogers MJ: The molecular mechanism of action of the anti-resorptive and anti-inflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis. Arthritis Rheum 2001, 44:2201-2210.
5.
Lehenkari PP, Kellinsalmi M, Napankangas JP, Ylitalo KV, Monkkonen J, Rogers MJ, Azhayev A, Vaananen HK, Hassinen IE: Further insight into mechanism of action of clodronate: inhibition of mitochondrial ADP/ATP translocase by a nonhydrolyzable, adenine-containing metabolite. Mol Pharmacol 2002, 61:1255-1262.
6.
van Beek E, Pieterman E, Cohen L, Lowik C, Papapoulos S: Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates. Biochem Biophys Res Commun 1999, 264:108-111.
7.
Bergstrom JD, Bostedor RG, Masarachia PJ, Reszka AA, Rodan G: Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase. Arch Biochem Biophys 2000, 373:231-241.
8.
Dunford JE, Thompson K, Coxon FP, Luckman SP, Hahn FM, Poulter CD, Ebetino FH, Rogers MJ: Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogencontaining bisphosphonates. J Pharmacol Exp Ther 2001, 296:235-242.
9.
Nancollas GH, Tang R, Phipps RJ, Henneman Z, Gulde S, Wu W, Mangood A, Russell RG, Ebetino FH: Novel insights into actions of bisphosphonates on bone: Differences in interactions with hydroxyapatite. Bone 2006, in press.
10. Leu CT, Luegmayr E, Freedman LP, Rodan GA, Reszka AA: Relative binding affinities of bisphosphonates for human bone and relationship to antiresorptive efficacy. Bone 2006, in press.
FPP synthase is the major pharmacological target of NBPs, accounting for not only the antiresorptive activity of these drugs, but also their ability to induce an acute-phase reaction, via the activation of gd T cells. Recent studies have clarified the exact mechanism by which N-BPs inhibit this enzyme, thus finally explaining the relationship between chemical structure and antiresorptive potency of these blockbuster drugs. However, further studies are needed to understand the subsequent cellular events in osteoclasts, as it remains unclear whether it is the loss of prenylated small GTPases or the parallel accumulation of unprenylated, mislocalized small GTPases that accounts for the inhibitory effects of NBPs on osteoclast function and survival.
11. Ebetino FH, Francis MD, Rogers MJ, Russell RGG: Mechanisms of action of etidronate and other bisphosphonates. Rev Contemp Pharmacother 1998, 9:233-243.
References and recommended reading
16. Coxon FP, Ebetino FH, Mules EH, Seabra MC, McKenna CE, Rogers MJ: Phosphonocarboxylate inhibitors of Rab geranylgeranyl transferase disrupt the prenylation and membrane localization of Rab proteins in osteoclasts in vitro and in vivo. Bone 2005, 37:349-358.
Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest 1.
Rogers MJ: New insights into the molecular mechanisms of action of bisphosphonates. Curr Pharm Des 2003, 9:2643-2658.
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12. Thompson K, Rogers MJ, Coxon FP, Crockett JC: Cytosolic entry of bisphosphonate drugs requires acidification of vesicles following fluid-phase endocytosis. Mol Pharmacol 2006, in press. 13. Frith JC, Rogers MJ: Antagonistic effects of different classes of bisphosphonates in osteoclasts and macrophages in vitro. J Bone Miner Res 2003, 18:204-212. 14. Takami M, Suda K, Sahara T, Itoh K, Nagai K, Sasaki T, Udagawa N, Takahashi N: Involvement of vacuolar H(+)-ATPase in incorporation of risedronate into osteoclasts. Bone 2003, 32:341-349. 15. Staal A, Frith JC, French MH, Swartz J, Gungor T, Harrity TW, Tamasi J, Rogers MJ, Feyen JH: The ability of statins to inhibit bone resorption is directly related to their inhibitory effect on HMG-CoA reductase activity. J Bone Miner Res 2003, 18:88-96.
17. Schindeler A, Little DG: Osteoclasts but not osteoblasts are affected by a calcified surface treated with zoledronic acid in vitro. Biochem Biophys Res Commun 2005, 338:710-716. 18. Rondeau JM, Bitsch F, Geiser M, Hemmig R, Kroemer M, Lehmann S, Ramage P, Rieffel S, Strauss A, Green JR, Jahnke W: Current Opinion in Pharmacology 2006, 6:307–312
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Structural basis for the exceptional in vivo efficacy of bisphosphonate drugs. ChemMedChem 2006, 1:267-273. See annotation to [19]. 19. Kavanagh K, Guo K, Dunford JE, Wu X, Knapp S, Ebetino FH, Rogers MJ, Russell RGG, Oppermann U: The molecular mechanism of nitrogen-containing bisphosphonates as anti-osteoporosis drugs: crystal structure and inhibition of farnesyl pyrophosphate synthase. Proc Natl Acad Sci U S A 2006, in press. The authors of this and the previous article reported, independently of each other, the first X-ray crystal structure of human FPP synthase and identified the mode of inhibition of the enzyme by N-BPs and residues crucial for drug–protein interactions. 20. Martin MB, Arnold W, Heath HT, Urbina JA, Oldfield E: Nitrogen-containing bisphosphonates as carbocation transition state analogs for isoprenoid biosynthesis. Biochem Biophys Res Commun 1999, 263:754-758. 21. Hosfield DJ, Zhang Y, Dougan DR, Brooun A, Tari LW, Swanson RV, Finn J: Structural basis for bisphosphonatemediated inhibition of isoprenoid biosynthesis. J Biol Chem 2003, 279:8526-8529. 22. Ebetino FH, Roze CN, McKenna CE, Barnett BL, Dunford JE, Russell RGG, Mieling GE, Rogers MJ: Molecular interactions of nitrogen-containing bisphosphonates within farnesyl diphosphate synthase. J Organomet Chem 2005, 690:2679-2687. 23. Thompson K, Rojas-Navea J, Rogers MJ: Alkylamines cause Vg9Vd2 T-cell activation and proliferation by inhibiting the mevalonate pathway. Blood 2006, 107:651-654. 24. Szabo CM, Matsumura Y, Fukura S, Martin MB, Sanders JM, Sengupta S, Cieslak JA, Loftus TC, Lea CR, Lee HJ et al.: Inhibition of geranylgeranyl diphosphate synthase by bisphosphonates and diphosphates: a potential route to new bone antiresorption and antiparasitic agents. J Med Chem 2002, 45:2185-2196.
32. Lerner EC, Qian Y, Blaskovich MA, Fossum RD, Vogt A, Sun J, Cox AD, Der CJ, Hamilton AD, Sebti SM: Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes. J Biol Chem 1995, 270:26802-26806. 33. Green JR: Bisphosphonates: preclinical review. Oncologist 2004, 9(Suppl 4):3-13. 34. Monkkonen H, Auriola S, Lehenkari P, Kellinsalmi M, Hassinen IE, Vepsalainen J, Monkkonen J: A new endogenous ATP analog (ApppI) inhibits the mitochondrial adenine nucleotide translocase (ANT) and is responsible for the apoptosis induced by nitrogen-containing bisphosphonates. Br J Pharmacol 2006, 147:437-445. 35. Fisher JE, Rogers MJ, Halasy JM, Luckman SP, Hughes DE, Masarachia PJ, Wesolowski G, Russell RG, Rodan GA, Reszka AA: 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 1999, 96:133-138. 36. Halasy-Nagy JM, Rodan GA, Reszka AA: Inhibition of bone resorption by alendronate and risedronate does not require osteoclast apoptosis. Bone 2001, 29:553-559. 37. van Beek ER, Cohen LH, Leroy IM, Ebetino FH, Lowik CW, Papapoulos SE: Differentiating the mechanisms of antiresorptive action of nitrogen containing bisphosphonates. Bone 2003, 33:805-811. 38. Adami S, Bhalla AK, Dorizzi R, Montesanti F, Rosini S, Salvagno G, Lo Cascio V: The acute-phase response after bisphosphonate administration. Calcif Tissue Int 1987, 41:326-331. 39. Kunzmann V, Bauer E, Wilhelm M: Gamma/delta T-cell stimulation by pamidronate. N Engl J Med 1999, 340:737-738.
25. Coxon FP, Rogers MJ: The role of prenylated small GTPbinding proteins in the regulation of osteoclast function. Calcif Tissue Int 2003, 72:80-84.
40. Thompson K, Rogers MJ: Statins prevent bisphosphonate induced g,d-T-cell proliferation and activation in vitro. J Bone Miner Res 2004, 19:278-288. This study demonstrate that N-BPs activate gd T cells indirectly through inhibition of FPP synthase and that statins can prevent this stimulatory effect of N-BPs on gd T cells in vitro.
26. Coxon FP, Helfrich MH, Larijani B, Muzylak M, Dunford JE, Marshall D, McKinnon AD, Nesbitt SA, Horton MA, Seabra MC et al.: Identification of a novel phosphonocarboxylate inhibitor of Rab geranylgeranyl transferase that specifically prevents Rab prenylation in osteoclasts and macrophages. J Biol Chem 2001, 276:48213-48222.
41. Gober HJ, Kistowska M, Angman L, Jeno P, Mori L, De Libero G: Human T cell receptor gd cells recognize endogenous mevalonate metabolites in tumor cells. J Exp Med 2003, 197:163-168. The authors show that Vg9Vd2 T cells recognize endogenous mevalonate metabolites that accumulate in N-BP-treated tumour cells in vitro.
27. Dunford JE, Rogers MJ, Ebetino FH, Phipps RJ, Coxon FP: Inhibition of protein prenylation by bisphosphonates causes sustained activation of Rac, Cdc42 and Rho GTPases. J Bone Miner Res 2006, in press. The authors demonstrate that N-BPs cause accumulation of active unprenylated Rho family GTPases in osteoclasts, raising the possibility that N-BPs might act by inappropriate stimulation, rather than inhibition, of small GTPase-dependent signalling pathways.
42. Sanders JM, Ghosh S, Chan JM, Meints G, Wang H, Raker AM, Song Y, Colantino A, Burzynska A, Kafarski P et al.: Quantitative structure-activity relationships for gd T cell activation by bisphosphonates. J Med Chem 2004, 47:375-384.
28. Allal C, Favre G, Couderc B, Salicio S, Sixou S, Hamilton AD, Sebti SM, Lajoie-Mazenc I, Pradines A: RhoA prenylation is required for promotion of cell growth and transformation and cytoskeleton organization but not for induction of serum response element transcription. J Biol Chem 2000, 275:31001-31008. 29. Khwaja A, Dockrell ME, Hendry BM, Sharpe CC: Prenylation is not necessary for endogenous Ras activation in nonmalignant cells. J Cell Biochem 2006, 97:412-422. 30. Vecchione C, Brandes RP: Withdrawal of 3-hydroxy-3methylglutaryl coenzyme A reductase inhibitors elicits oxidative stress and induces endothelial dysfunction in mice. Circ Res 2002, 91:173-179. 31. Cordle A, Koenigsknecht-Talboo J, Wilkinson B, Limpert A, Landreth G: Mechanisms of statin-mediated inhibition of small G-protein function. J Biol Chem 2005, 280:34202-34209.
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43. Hewitt RE, Lissina A, Green AE, Slay ES, Price DA, Sewell AK: The bisphosphonate acute phase response: rapid and copious production of proinflammatory cytokines by peripheral blood gd T cells in response to aminobisphosphonates is inhibited by statins. Clin Exp Immunol 2005, 139:101-111. 44. Boissier S, Ferreras M, Peyruchaud O, Magnetto S, Ebetino FH, Colombel M, Delmas P, Delaisse JM, Clezardin P: Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases. Cancer Res 2000, 60:2949-2954. 45. Roelofs AJ, Hulley PA, Meijer A, Ebetino FH, Russell RGG, Shipman CM: Selective inhibition of Rab prenylation by a phosphonocarboxylate analogue of risedronate induces apoptosis, but not S-phase arrest, in human myeloma cells. Int J Cancer 2006, in press. 46. Lackner MR, Kindt RM, Carroll PM, Brown K, Cancilla MR, Chen C, de Silva H, Franke Y, Guan B, Heuer T et al.: Chemical genetics identifies Rab geranylgeranyl transferase as an apoptotic target of farnesyl transferase inhibitors. Cancer Cell 2005, 7:325-336.
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