F P Coxon

Nitrogen-Containing Bisphosphonates Inhibit the Mevalonate Pathway and Prevent Post-Translational Prenylation of GTP-Binding Proteins, Including Ras

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of metabolic bone diseases. Although the molecular targets of bisphosphonates have not been identified, these compounds inhibit bone resorption by mechanisms that can lead to osteoclast apoptosis. Bisphosphonates also induce apoptosis in mouse J774 macrophages in vitro, probably by the same mechanisms that lead to osteoclast apoptosis. We have found that, in J774 macrophages, nitrogen‐containing bisphosphonates (such as alendronate, ibandronate, and risedronate) inhibit post‐translational modification (prenylation) of proteins, including the GTP‐binding protein Ras, with farnesyl or geranylgeranyl isoprenoid groups. Clodronate did not inhibit protein prenylation. Mevastatin, an inhibitor of 3‐hydroxy‐3‐methylglutatyl (HMG)‐CoA reductase and hence the biosynthetic pathway required for the production of farnesyl pyrophosphate and geranylgeranyl pyrophosphate, also caused apoptosis in J774 macrophages and murine osteoclasts in vitro. Furthermore, alendronate‐induced apoptosis, like mevastatin‐induced apoptosis, could be suppressed in J774 cells by the addition of farnesyl pyrophosphate or geranylgeranyl pyrophosphate, while the effect of alendronate on osteoclast number and bone resorption in murine calvariae in vitro could be overcome by the addition of mevalonic acid. These observations suggest that nitrogen‐containing bisphosphonate drugs cause apoptosis following inhibition of post‐translational prenylation of proteins such as Ras. It is likely that these potent antiresorptive bisphosphonates also inhibit bone resorption by preventing protein prenylation in osteoclasts and that enzymes of the mevalonate pathway or prenyl protein transferases are the molecular targets of the nitrogen‐containing bisphosphonates. Furthermore, the data support the view that clodronate acts by a different mechanism.

Heterocycle‐Containing Bisphosphonates Cause Apoptosis and Inhibit Bone Resorption by Preventing Protein Prenylation: Evidence from Structure‐Activity Relationships in J774 Macrophages

Recent evidence suggests that bisphosphonates (BPs) may inhibit bone resorption by mechanisms that lead to osteoclast apoptosis. We have previously shown that BPs also reduce cell viability and induce apoptosis in the macrophage‐like cell line J774. To determine whether BPs inhibit osteoclast‐mediated bone resorption and affect J774 macrophages by the same molecular mechanism, we examined the potency to reduce J774 cell viability of pairs of nitrogen‐containing BPs that differ slightly in the structure of the heterocycle‐containing side chain but that differ markedly in antiresorptive potency. In all cases, the most potent antiresorptive BP of each pair also caused the greatest loss of J774 viability, while the less potent antiresorptive BPs were also less potent at reducing J774 cell viability. Similarly, the bisphosphinate, phosphonoalkylphosphinate and monophosphonate analogs of BPs (in which one or both phosphonate groups are modified, giving rise to much less potent or inactive antiresorptive agents) were much less potent or inactive at reducing J774 cell viability. Thus, the structure‐activity relationships of BPs for inhibiting bone resorption match those for causing loss of cell viability in J774 cells, indicating that BPs inhibit osteoclast‐mediated bone resorption and reduce J774 macrophage viability by the same molecular mechanism. Loss of J774 cell viability after treatment with BPs was associated with a parallel increase in apoptotic cell death. We have recently proposed that nitrogen‐containing BPs reduce cell viability and cause J774 apoptosis as a consequence of inhibition of enzymes of the mevalonate pathway and hence loss of prenylated proteins. In this study, the BPs that were potent inducers of J774 apoptosis and potent antiresorptive agents were also found to be effective inhibitors of protein prenylation in J774 macrophages, whereas the less potent BP analogs did not inhibit protein prenylation. This provides strong evidence that BPs with a heterocyclic, nitrogen‐containing side chain, such as risedronate, inhibit osteoclast‐mediated bone resorption and induce J774 apoptosis by preventing protein prenylation.

Molecular mechanisms of action of bisphosphonates.

BPs can be grouped into two general classes according to their chemical structure and the molecular mechanism by which they inhibit osteoclast-mediated bone resorption. The simple BPs can be metabolically incorporated into non-hydrolysable analogues of ATP that accumulate intracellularly in osteoclasts, causing osteoclast cell death by apoptosis. By contrast, the more potent N-BPs inhibit FPP synthase, an enzyme in the mevalonate pathway. Inhibition of this enzyme in osteoclasts prevents the biosynthesis of isoprenoid lipids that are required for the prenylation of small GTPase signalling proteins necessary for osteoclast function. Inhibition of FPP synthase in cells other than osteoclasts also appears to account for the adverse effects of N-BPs in vivo (including the acute phase reaction) and for the anti-tumour effects of N-BPs in vitro.

Identification of a Novel Phosphonocarboxylate Inhibitor of Rab Geranylgeranyl Transferase That Specifically Prevents Rab Prenylation in Osteoclasts and Macrophages

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

Fluorescently Labeled Risedronate and Related Analogues: “Magic Linker” Synthesis

We report synthesis of the first fluorescently labeled conjugates of risedronate (1), using an epoxide linker strategy enabling conjugation of 1 via its pyridyl nitrogen with the label (carboxyfluorescein). Unlike prior approaches to create fluorescent bisphosphonate probes, the new linking chemistry did not abolish the ability to inhibit protein prenylation in vitro, while significantly retaining hydroxyapatite affinity. The utility of a fluorescent 1 conjugate in visualizing osteoclast resorption in vitro was demonstrated.