Further Insight into Mechanism of Action of Clodronate: Inhibition of Mitochondrial ADP/ATP Translocase by a Nonhydrolyzable, Adenine-Containing Metabolite

Abstract

Bisphosphonates are currently the most important class of antiresorptive drugs used for the treatment of diseases with excess bone resorption. Recent studies have shown that bisphosphonates can be divided into two groups with distinct molecular mechanisms of action depending on the nature of the R 2 side chain. Alendronate, like other nitrogen-containing bisphosphonates, inhibits bone resorption and causes apoptosis of osteoclasts and other cells in vitro by preventing post-translational modification of GTP-binding proteins with isoprenoid lipids. Clodronate, a bisphosphonate that lacks a nitrogen, does not inhibit protein isoprenylation but can be metabolized intracellularly to a ␤-␥-methylene (AppCp-type) analog of ATP, which is cytotoxic to macrophages in vitro. The detailed molecular basis for the cytotoxic effects of adenosine-5Ј-[␤,␥-dichloromethylene]triphosphate (AppCCl 2 p) has not been determined yet. We addressed this question by studying the effects of alendronate, clodronate, and the clodronate metabolite AppCCl 2 p on isolated mitochondria, mitochondrial fractions, and mitochondrial membrane potential in isolated human osteoclasts. We found that AppCCl 2 p inhibits mitochondrial oxygen consumption by a mechanism that involves competitive inhibition of the ADP/ATP translocase. Alendronate or the native form of clodronate did not have any immediate effect on mitochondria. However, longer treatment with liposome-encapsulated clodronate caused collapse of the mitochondrial membrane potential, although prominent apoptosis was a late event. Hence, inhibition of the ADP/ATP translocase by the metabolite AppCCl 2 p is a likely route by which clodronate causes osteoclast apoptosis and inhibits bone resorption.

The kd/kd Mouse Is a Model of Collapsing Glomerulopathy

“…If this is indeed correct, B6 kd/kd mice would represent the first model of CG due to a mitochondrial disorder, providing a ready system to investigate how environmental factors may influence the manifestation of this abnormality within podocytes. Interestingly, bisphosphonate drugs, small molecules linked to podocyte injury and CG in humans (24,25), can perturb mitochondrial function (26), and human mitochondrial transprenyltransferases sharing homology with PLMP contain specificity determining residues for bisphosphonate binding (i.e., the amino acid sequence DDXXD). However, the extent to which bisphosphonates interact with and inhibit human transprenyltransferases is still unclear (Eric Oldfield, University of Illinois at Urbana-Champaign, personal communication).…”

Bisphosphonates: Mode of Action and Pharmacology

“…The AppCp-type metabolites of bisphosphonates are cytotoxic when internalized and cause similar changes in morphology to those observed in clodronate-treated cells, possibly by interference with mitochondrial ATP translocases. 64 Overall, this group of bisphosphonates, therefore, seem to act as prodrugs, being converted to active metabolites after intracellular uptake by osteoclasts in vivo.…”

Bisphosphonates: Preclinical Review

“…First-generation, nonnitrogen-containing bisphosphonates are metabolized by osteoclasts to nonhydrolyzable cytotoxic ATP analogues [18][19][20][21]. For example, clodronate is metabolized to AppCC12p, which, at high concentrations, inhibits mitochondrial ATP/adenosine diphosphate (ADP) translocase, thereby causing loss of the mitochondrial membrane potential and direct induction of apoptosis [22][23][24]. The high affinity of bisphosphonates for bone mineral and subsequent uptake by activated osteoclasts during bone resorption ensures that cytotoxic concentrations of these metabolites only accumulate within osteoclasts.…”