The bisphosphonate tiludronate is a potent inhibitor of the osteoclast vacuolar H+-ATPase

Abstract

Although bisphosphonates have been shown to be potent inhibitors of osteoclast-mediated bone resorption in vivo and in vitro and are used as therapeutic agents in hyper-resorptive bone diseases such as Paget disease or hypercalcemia of malignancy, their exact biochemical target(s) and mode(s) of action are for the most part still unknown. The resorption of bone requires solubilization of the mineral component of the matrix, achieved by acidification of the resorbing compartment by a vacuolar-type proton ATPase (V-ATPase) present in the ruffled border membrane of osteoclasts. Since we have shown that the V-ATPase is inhibited by both ADP and phosphate, which share structural characteristics with bisphosphonates, we hypothesized that inhibition of the osteoclast V-ATPase could be one of the mechanism(s) by which bisphosphonates inhibit bone resorption. Pyrophosphate and the bisphosphonates etidronate, alendronate, and YM-175 inhibited proton transport in membrane vesicles derived from chicken kidney and osteoclasts but with very low potency (IC50 > or = 5 mM). In contrast, the ability of tiludronate to inhibit proton transport was 5-fold higher in kidney-derived vesicles (IC50 = 1.1 mM) and 10,000-fold higher in vesicles derived from osteoclasts (IC50 = 466 nM). Tiludronate also potently inhibited proton transport in yeast microsomal preparations (IC50 = 3.5 microM) and inhibited the activity of purified yeast V-ATPase. The inhibition of the osteoclast V-ATPase-mediated proton transport by tiludronate was rapid, pH-dependent, and reversible. No change in membrane vesicle permeability to protons was detected. The inhibition was noncompetitive with respect to ATP, and tiludronate did not protect the pump from inactivation by N-ethylmaleimide, strongly suggesting that tiludronate does not bind to the catalytic site of the enzyme. It is concluded that tiludronate is a significantly more potent inhibitor of V-ATPase than other bisphosphonates and that it has a significant degree of selectivity for the avian osteoclast V-ATPase relative to the avian kidney V-ATPase.

Zoledronate induces apoptosis in cells from fibro‐cellular membrane of unicameral bone cyst (UBC)

“…Nitrogen containing bisphosphonates, such as zoledronate, demonstrate (1) inhibition of osteoclastogenesis; (2) protection of host bone from osteoclastic resorption and (3) induction of apoptosis of osteoclasts in osteoporosis, metastatic bone cancers, Paget's disease, and benign bone tumor-like lesions [7,10,16,27,30,32,33]. Interestingly, even though UBC and trabecular bone cells in vitro shared similar chemokine/cytokine expression according to ICC staining, UBC cells seemed to be more susceptible to both bisphosphonates and GGOH than trabecular bone cells.…”

“…Bisphosphonates are widely used anti-resorptive agents that effectively inhibit osteoclastogenesis, inactivate and induce apoptosis of osteoclasts in osteoporosis, metastatic bone cancers, osteogenesis imperfecta, Paget's disease and multi-focal benign bone tumor-like lesions such as polyostotic fibrous dysplasia [7,10,16,27,30,32,33]. Bisphosphonates are ideally suited for the treatment of some bone diseases because they bind avidly to the bone mineral and thus accumulate in bone at sites of active bone metabolism.…”

The development of bisphosphonates for therapeutic uses, and bisphosphonate structure-activity consideration

“…[22][23][24][25][26] The effect on the osteoclast leads to a decrease in bone turnover, and is secondary to the inhibition of bone resorption. Several studies 22,24,[27][28][29][30] indicate that BPs can be classified into at least two groups, with different modes of action. BPs that most closely resemble PPi (such as Clodronate and Etidronate, Figure 3) can be metabolically incorporated into non-hydrolyzable analogues of ATP-dependent intracellular enzymes.…”

Bisphosphonates

“…The vacuolar type proton ATPase appears to be responsible in part for this extrusion. When this proton pump was assayed in insideout osteoclast vesicles in vitro, tiludronate was found to be a potent inhibitor (48), while other bisphosphonates more potent in vivo were not inhibitory. However, it should be emphasized again that the various bisphosphonates do not necessarily have a common mechanism of action, especially at the molecular level.…”