J E Dunford

The molecular mechanism of nitrogen-containing bisphosphonates as antiosteoporosis drugs.

Osteoporosis and low bone mass are currently estimated to be a major public health risk affecting >50% of the female population over the age of 50. Because of their bone-selective pharmacokinetics, nitrogen-containing bisphosphonates (N-BPs), currently used as clinical inhibitors of bone-resorption diseases, target osteoclast farnesyl pyrophosphate synthase (FPPS) and inhibit protein prenylation. FPPS, a key branchpoint of the mevalonate pathway, catalyzes the successive condensation of isopentenyl pyrophosphate with dimethylallyl pyrophosphate and geranyl pyrophosphate. To understand the molecular events involved in inhibition of FPPS by N-BPs, we used protein crystallography, enzyme kinetics, and isothermal titration calorimetry. We report here high-resolution x-ray structures of the human enzyme in complexes with risedronate and zoledronate, two of the leading N-BPs in clinical use. These agents bind to the dimethylallyl/geranyl pyrophosphate ligand pocket and induce a conformational change. The interactions of the N-BP cyclic nitrogen with Thr-201 and Lys-200 suggest that these inhibitors achieve potency by positioning their nitrogen in the proposed carbocation-binding site. Kinetic analyses reveal that inhibition is competitive with geranyl pyrophosphate and is of a slow, tight binding character, indicating that isomerization of an initial enzyme–inhibitor complex occurs with inhibitor binding. Isothermal titration calorimetry indicates that binding of N-BPs to the apoenzyme is entropy-driven, presumably through desolvation entropy effects. These experiments reveal the molecular binding characteristics of an important pharmacological target and provide a route for further optimization of these important drugs.

Inhibition of protein prenylation by bisphosphonates causes sustained activation of Rac, Cdc42, and Rho GTPases

N‐BPs, which inhibit bone resorption by preventing prenylation of small GTPases, unexpectedly cause the accumulation of GTP‐bound, unprenylated Rho family GTPases in macrophages and osteoclasts. In macrophages, this also leads to sustained, Rac‐mediated activation of p38. The antiresorptive activity of N‐BPs may therefore be caused at least in part, by the accumulation of unprenylated small GTPases, causing inappropriate activation of downstream signaling pathways.

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.

Identification of Biphenylcarboxylic Acid Derivatives as a Novel Class of Bone Resorption Inhibitors

A novel class of biphenylcarboxylic acid derivatives are described that inhibit osteoclastic bone resorption in vitro by promoting osteoclast apoptosis and that prevent ovariectomy‐induced bone loss in vivo. The compounds act by a novel mechanism that seems to be distinct from existing antiresorptive drugs.