Metabolism of halogenated bisphosphonates by the cellular slime mould dictyostelium discoideum

doi:10.1016/0006-291X(92)91574-A

Biochemical and Biophysical Research Communications

Volume 189, Issue 1, 30 November 1992, Pages 414-423

https://doi.org/10.1016/0006-291X(92)91574-A Get rights and content

Abstract

Methylenebisphosphonate and its monofluoro-, difluoro- and dichloro- derivatives inhibited growth of amoebae of Dictyostelium discoideum. Dichloromethylenebisphosphonate was the most potent inhibitor of amoebal growth whereas difluoromethylenebisphosphonate was the least potent inhibitor. Each of the bisphosphonate was taken up by the amoebae and incorporated into the corresponding β,γ-methylene analogue of adenosine triphosphate. Two of the bisphosphonates were also incorporated into the corresponding analogues of diadenosyl tetraphosphate. No correlation was found between the ability of the bisphosphonates to inhibit amoebal growth and the extent to which they were metabolised.

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Bisphosphonates (BPs) are a class of bone-seeking agents that are selectively internalized by osteoclasts and inhibit bone resorption via two distinct mechanisms. Whereas BPs of simple chemical structure are incorporated into toxic, nonhydrolyzable adenosine triphosphate analogues, the more potent, nitrogen-containing BPs (N-BPs) inhibit farnesyl pyrophosphate synthase, an enzyme of the mevalonate–cholesterol biosynthesis pathway. This disrupts the production of isoprenoid lipids and prevents the prenylation of small GTPase proteins necessary for osteoclast function, and causes accumulation of a toxic, isoprenoid-containing metabolite (ApppI). Although additional molecular targets for BPs may exist, these two main mechanisms of action account for the antiresorptive effects of these agents on osteoclasts, as well as for some adverse effects such as the acute phase response to N-BPs. Further studies are required to fully understand the (perhaps subtle) effects in vivo of BPs on other cell types in bone such as osteocytes. The mechanisms underlying the antitumor actions of N-BPs in mouse models, and apparent beneficial effects outside the skeleton in cancer patients, remain unclear. Increasing evidence suggests that these effects occur via immune myeloid cells such as tumor-associated macrophages, thereby indirectly decreasing tumor growth and metastasis.
Molecular mechanisms of action of bisphosphonates and new insights into their effects outside the skeleton
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Citation Excerpt :
The discovery of how simple BPs are toxic to cells came about from work using methylene-bisphosphonate (medronate) to study intracellular pH in amoebae of the slime mould Dictyostelium [28], which internalise BPs in solution by endocytosis (like osteoclasts) [29]. Following cellular uptake, Dictyostelium amoebae were found to metabolise medronate and other simple BPs into a class of ATP (adenosine triphosphate) analogues that contain a methylene group between the β and γ phosphate residues (β-γ methylene-containing, AppCp-type analogues of ATP) (Fig. 3A), as well as analogues of diadenosine tetraphosphate (AppCppA analogues of AP4A) [28,30–32]. This observation brought together Graham Russell, Donald Watts, Michael Blackburn, Hal Ebetino and PhD student Mike Rogers.
Bisphosphonates (BP) are a class of calcium-binding drug used to prevent bone resorption in skeletal disorders such as osteoporosis and metastatic bone disease. They act by selectively targeting bone-resorbing osteoclasts and can be grouped into two classes depending on their intracellular mechanisms of action. Simple BPs cause osteoclast apoptosis after cytoplasmic conversion into toxic ATP analogues. In contrast, nitrogen-containing BPs potently inhibit FPP synthase, an enzyme of the mevalonate (cholesterol biosynthesis) pathway. This results in production of a toxic metabolite (ApppI) and the loss of long-chain isoprenoid lipids required for protein prenylation, a process necessary for the function of small GTPase proteins essential for the survival and activity of osteoclasts. In this review we provide a state-of-the-art overview of these mechanisms of action and a historical perspective of how they were discovered. Finally, we challenge the long-held dogma that BPs act only in the skeleton and highlight recent studies that reveal insights into hitherto unknown effects on tumour-associated and tissue-resident macrophages.
Bisphosphonates—much more than only drugs for bone diseases
2020, European Journal of Pharmacology
Citation Excerpt :
At the molecular level, the biochemical mechanisms of action of BPs differ depending on their structure, which is the basis for their classification into at least two groups (Fig. 3). The simpler nitrogen-free BPs, such as clodronate and etidronate, behave as pyrophosphate analogs by getting metabolically incorporated into the stable analogs of ATP through the action of aminoacyl-tRNA synthase (Rogers et al., 1996, 1992). The intracellular accumulation of these nonhydrolyzable metabolites within osteoclasts causes a deficiency of functional ATPs, as well as inhibits the mitochondrial ADP/ATP translocase, leading to the apoptosis of osteoclasts (Frith et al., 2001; Lehenkari et al., 2002).
α,α-Bisphosphonates (BPs) are well established in the treatment of bone diseases such as osteoporosis and Paget's disease. Their successful application originates from their high affinity to hydroxyapatite. While the initially appreciated features of BPs are already beneficial to many patients, recent developments have further expanded their pleiotropic applications. This review describes the background of the interactions of BPs with bone cells that form the basis of the classical treatment.
A better understanding of the mechanism behind their interactions allows for the parallel application of BPs against bone cancer and metastases followed by palliative pain relief. Targeted therapy with bone-seeking BPs coupled with a diagnostic agent in one particle resulted in theranostics which is also described here. For example, in such a system, BP moieties are bound to contrast agents used in magnetic resonance imaging or radionuclides used in positron emission tomography. In addition, another example of the pleiotropic function of BPs which involves targeting the imaging agents to bone tissues accompanied by pain reduction is presented in this work.
Activation of microglia in acute hippocampal slices affects activity-dependent long-term potentiation and synaptic tagging and capture in area CA1
2019, Neurobiology of Learning and Memory
Activity dependent setting of synaptic tags is critical for the establishment and maintenance of long-term plasticity and its associative properties such as synaptic tagging and capture (STC), a widely studied cellular model of associative memory. Although the known mechanisms of STC such as setting of synaptic tags or distribution of plasticity related proteins (PRPs) are the processes mainly happening within the neuronal compartments, the role of non-neuronal components is still elusive. Here, we report that microglia has a specific role in setting the synaptic tags and thus promotes long-term plasticity and STC. Treatment of hippocampal slices with clodronate, a specific inhibitor of microglia, resulted in an activated morphology of microglia but not of the hippocampal pyramidal neurons, oligodendrocytes or astrocytes. Activation of microglia before or 60 min after the induction of long-term plasticity prevented its maintenance and thus the expression of STC. Interestingly, activation of microglia 2 h after the induction of long-term plasticity neither prevented its maintenance nor its associative interaction with activated nearby synaptic populations. Given the half-life of synaptic tags is until about 60–90 min, activation of microglia beyond this time point while the maintenance phase is still unperturbed, suggests a lack of microglial interference in the synthesis or trigger of plasticity related products. Thus, our study provides the first evidence that microglia play a critical role in the setting of synaptic tags during the early phase of activity dependent plasticity.
Cellular and molecular actions of bisphosphonates
2015, Bone Cancer: Primary Bone Cancers and Bone Metastases: Second Edition
Bisphosphonates (BPs) are a class of bone-seeking agents that are selectively internalized by osteoclasts and inhibit bone resorption via two distinct mechanisms. Whereas BPs of simple chemical structure are incorporated into toxic, non-hydrolyzable ATP analogs, the more potent, nitrogen-containing BPs (N-BPs) inhibit FPP synthase, an enzyme of the cholesterol biosynthetic (mevalonate) pathway. This disrupts the production of isoprenoid lipids and prevents the prenylation of small GTPase proteins necessary for osteoclast function, and causes accumulation of a toxic, isoprenoid-containing metabolite (ApppI). Although additional molecular targets for BPs may exist, these two main mechanisms of action account for the anti-resorptive effects of these agents on osteoclasts, as well as for some adverse effects such as the acute phase response to N-BPs. Further studies are required to fully understand the (perhaps subtle) effects in vivo of BPs on other cell types such as osteocytes. The mechanisms underlying the anti-tumor actions of N-BPs in mouse models, and apparent beneficial effects outside the skeleton in cancer patients, remain unclear. Increasing evidence suggests that these effects occur via immune myeloid cells such as tumor-associated macrophages, thereby indirectly decreasing tumor growth and metastasis.
Biochemical and molecular mechanisms of action of bisphosphonates
2011, Bone
Citation Excerpt :
As a result, AppCH2p resembles ATP but is resistant to hydrolytic breakdown and release of phosphate. Although this initial study only showed that medronate could be metabolised, we [7,8] and others [9] soon found that clodronate and other BPs of simple chemical structure that closely resemble PPi, such as etidronate, could also be metabolised by Dictyostelium amoebae to methylene-containing (AppCp-type) analogues of ATP (Fig. 2). Furthermore, the accumulation of AppCp-type metabolites of BPs was associated with cytotoxicity and inhibition of proliferation of Dictyostelium amoebae [6,8–10].
This review describes the key discoveries over the last 15 years that have led to a clearer understanding of the molecular mechanisms by which bisphosphonate drugs inhibit bone resorption. Once released from bone mineral surfaces during bone resorption, these agents accumulate intracellularly in osteoclasts. Simple bisphosphonates such as clodronate are incorporated into non-hydrolysable analogues of adenosine triphosphate, which induce osteoclast apoptosis. The considerably more potent nitrogen-containing bisphosphonates are not metabolised but potently inhibit farnesyl pyrophosphate (FPP) synthase, a key enzyme of the mevalonate pathway. This prevents the synthesis of isoprenoid lipids necessary for the post-translational prenylation of small GTPases, thereby disrupting the subcellular localisation and normal function of these essential signalling proteins. Inhibition of FPP synthase also results in the accumulation of the upstream metabolite isopentenyl diphosphate, which is incorporated into the toxic nucleotide metabolite ApppI. Together, these properties explain the ability of bisphosphonate drugs to inhibit bone resorption by disrupting osteoclast function and survival. These discoveries are also giving insights into some of the adverse effects of bisphosphonates, such as the acute phase reaction that is triggered by inhibition of FPP synthase in peripheral blood monocytes.
This article is part of a Special Issue entitled Bisphosphonates.

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Metabolism of halogenated bisphosphonates by the cellular slime mould dictyostelium discoideum

Abstract

Lancet

Lancet

Anal. Biochem

Chem. Industry (London)

J. Chem. Soc., Chem. Commun

Biochemistry

J. Clin. Invest

Progr. Basic Clin. Pharmacol

Nature