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M J Rogers

Publications and source records attributed to M J Rogers.

At least 19 recordsLinked to original sources
PubMed2004

From molds and macrophages to mevalonate: a decade of progress in understanding the molecular mode of action of bisphosphonates.

Although bisphosphonates were first used as therapeutic agents to inhibit bone resorption in the early 1970s, their mode of action at the molecular level has only become fully clear within the last few years. One of the reasons for this lack of understanding was the difficulty in isolating large numbers of pure osteoclasts for biochemical studies. In the last decade, the identification of appropriate surrogate models that reflected the antiresorptive potencies of bisphosphonates, such as Dictyostelium slime molds and macrophages, helped overcome this problem and proved to be instrumental in elucidating the molecular pathways by which these compounds inhibit osteoclast-mediated bone resorption. This brief review summarizes our current understanding of these pathways.

Animals↗
PubMed2002

The role of prenylated small GTP-binding proteins in the regulation of osteoclast function.

The Ras superfamily of small GTP-binding proteins (also known as small GTPases) comprises more than 80 highly conserved proteins of the Ras, Rho, and Rab subfamilies that are involved in multiple intracellular signalling pathways. These proteins are able to function as molecular switches in the transduction of signals from membrane receptors by cycling between an inactive, GDP-bound state and an active, GTP-bound state, which can then interact with a number of different effector molecules (Fig. 1). The activity of small GTPases is regulated by three classes of regulatory proteins: guanine nucleotide exchange factors (GEFs), which catalyse the exchange of GDP for GTP, thereby activating the small GTPase; GTPase-activating proteins (GAPs), which enhance the intrinsic ability of small GTPases to hydrolyse GTP, resulting in reversion to the inactive GDP-bound state; and guanine nucleotide dissociation inhibitors (GDIs), which preferentially bind to the GDP-bound GTPases in the cytoplasm, thereby inhibiting the release of GDP and maintaining the GTPase in the inactive state [1]. GDIs have not been identified for all small GTPases, but play an important role in the control of the Rho family GTPases.

Animals↗
PubMed2001

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 approximately 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 [(3)H]geranylgeraniol. Furthermore, NE10790 inhibited incorporation of [(14)C]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.

Alkyl and Aryl Transferases↗
PubMed2001

The molecular mechanism of action of the antiresorptive and antiinflammatory drug clodronate: evidence for the formation in vivo of a metabolite that inhibits bone resorption and causes osteoclast and macrophage apoptosis.

OBJECTIVE: The primary aims of this study were to determine whether clodronate and liposome-encapsulated clodronate are metabolized to adenosine 5'-(beta,gamma-dichloromethylene) triphosphate (AppCCl2p) by osteoclasts and macrophages in vivo, and to determine whether intracellular accumulation of this metabolite accounts for the antiresorptive and antimacrophage effects of clodronate. To compare the mechanism of action of clodronate and alendronate, effects on protein prenylation in osteoclasts and macrophages in vivo were also assessed. METHODS: High-performance liquid chroma-tography-mass spectrometry was used to determine whether rabbit osteoclasts (purified ex vivo with immunomagnetic beads) metabolize clodronate, and whether rat peritoneal macrophages metabolize liposome-encapsulated clodronate, following in vivo administration. The effects of clodronate and AppCCl2p on bone resorption, osteoclast number, and apoptosis in vitro were compared. Using an antibody to the unprenylated form of RaplA, effects on protein prenylation were assessed by Western blot analysis of osteoclast and peritoneal macrophage lysates from bisphosphonate-treated animals. RESULTS: AppCCl2p could be detected in extracts from osteoclasts purified from clodronate-treated rabbits. Intracellular accumulation of AppCCl2p caused a reduction in the number of osteoclasts, increased osteoclast apoptosis, and inhibited bone resorption in vitro. These effects were indistinguishable from those of clodronate. Liposome-encapsulated clodronate was also metabolized to AppCCl2p by rat peritoneal macrophages in vivo. Liposome-encapsulated clodronate caused an increase in peritoneal macrophage apoptosis in ex vivo cultures that was indistinguishable from the increase in apoptosis caused by liposome-encapsulated AppCCl2p. Unlike alendronate, clodronate and its metabolite did not affect prenylation of the small GTPase RaplA in osteoclasts or macrophages in vivo. CONCLUSION: These results provide the first direct evidence that the antiinflammatory and antiresorptive effects of clodronate on macrophages and osteoclasts in vivo occur via the intracellular formation of AppCCl2p.

Adenosine Triphosphate↗
PubMed2001

Visualization of bisphosphonate-induced caspase-3 activity in apoptotic osteoclasts in vitro.

Bisphosphonates inhibit osteoclast-mediated bone resorption by mechanisms that have only recently become clear. Whereas nitrogen-containing bisphosphonates affect osteoclast function by preventing protein prenylation (especially geranylgeranylation), non-nitrogen-containing bisphosphonates have a different molecular mechanism of action. In this study, we demonstrate that nitrogen-containing bisphosphonates (risedronate, alendronate, pamidronate, and zoledronic acid) and non-nitrogen-containing bisphosphonates (clodronate and etidronate) cause apoptosis of rabbit osteoclasts, human osteoclastoma-derived osteoclasts, and human osteoclast-like cells generated in cultures of bone marrow in vitro. Osteoclast apoptosis was shown to involve characteristic morphological changes, loss of mitochondrial membrane potential, and the activation of caspase-3-like proteases capable of cleaving peptide substrates with the sequence DEVD. Caspase-3-like activity could be visualized in unfixed, dying osteoclasts and osteoclast-like cells using a cell-permeable, fluorogenic substrate. Bisphosphonate-induced osteoclast apoptosis was dependent on caspase activation, because apoptosis resulting from alendronate, clodronate, or zoledronic acid treatment was suppressed by zVAD-fmk, a broad-range caspase inhibitor, or by SB-281277, a specific isatin sulfonamide inhibitor of caspase-3/-7. Furthermore, caspase-3 (but not caspase-6 or caspase-7) activity could be detected and quantitated in lysates from purified rabbit osteoclasts, whereas the p17 fragment of active caspase-3 could be detected in human osteoclast-like cells by immunofluorescence staining. Caspase-3, therefore, appears to be the major effector caspase activated in osteoclasts by bisphosphonate treatment. Caspase activation and apoptosis induced by nitrogen-containing bisphosphonates are likely to be the consequence of the loss of geranylgeranylated rather than farnesylated proteins, because the ability to cause apoptosis and caspase activation was mimicked by GGTI-298, a specific inhibitor of protein geranylgeranylation, whereas FTI-277, a specific inhibitor of protein farnesylation, had no effect on apoptosis or caspase activity.

Animals↗
PubMed2001

Nitrogen-containing bisphosphonates induce apoptosis of Caco-2 cells in vitro by inhibiting the mevalonate pathway: a model of bisphosphonate-induced gastrointestinal toxicity.

Bisphosphonates have become an important addition to the pharmacological armamentarium against postmenopausal osteoporosis. One of the major side effects of oral therapy with some nitrogen-containing bisphosphonates appears to be gastrointestinal (GI) intolerability, particularly esophageal irritation and ulceration. Because nitrogen-containing bisphosphonates can cause apoptosis in a variety of cell types in vitro, by inhibiting the mevalonate pathway, we hypothesized that the effect of these agents on the GI tract may be due to apoptosis or inhibition of growth of gut epithelial cells. A comparison between clodronate, etidronate, pamidronate, alendronate, and risedronate demonstrated that only the nitrogen-containing bisphosphonates were effective at inducing apoptosis or inhibiting proliferation of Caco-2 human epithelial cells in vitro, at concentrations of between 10 and 1000 micromol/L. The ability of nitrogen-containing bisphosphonates to cause apoptosis and inhibit Caco-2 cell proliferation was due to inhibition of the mevalonate pathway, because the addition of farnesol, oxidized low-density lipoprotein (LDL) cholesterol, or especially geranylgeraniol suppressed the effects. Furthermore, pamidronate, alendronate, and risedronate inhibited protein prenylation in Caco-2 cells, as determined by analysis of the processing of Rap1A, a prenylated small GTPase. These studies suggest that the effects of nitrogen-containing bisphosphonates observed in the GI tract may be due to inhibition of proliferation or apoptosis of gut epithelial cells, following loss of prenylated proteins and sterols.

Apoptosis↗
PubMed2001

The cellular uptake and metabolism of clodronate in RAW 264 macrophages.

PURPOSE: Non-nitrogen-containing bisphosphonates, such as clodronate (dichloromethylene bisphosphonate), appear to act as prodrugs, their active form being the AppCp-type analogues of ATP. To further elucidate this, we examined the cellular uptake of clodronate and intracellular accumulation of the metabolite of clodronate (AppCCl2p) in RAW 264 macrophages, the influence of clodronate metabolism on the intracellular ATP concentration, and the time course of clodronate metabolism and the effects of clodronate on cytokine secretion from macrophages. METHODS: The cellular uptake of clodronate was measured using 14C-labeled clodronate. AppCCl2p was determined in cell extracts by using an ion-pairing HPLC-ESI-MS. The cytokine concentrations in the culture supernatants were measured with time-resolved fluoroimmunoassay. Intracellular ATP concentration was measured with a luminometer using a luciferin-luciferase assay. RESULTS: Of the clodronate internalized by macrophages in vitro, 30-55% is metabolized to AppCCl2p, which accumulates to high intracellular concentrations during the first 12 h of exposure. This accumulation does not affect the ATP levels in the cells. The time course of metabolite appearance in the cells and the inhibition of cytokine secretion were very similar. CONCLUSIONS: These results strongly support the idea that clodronate acts as a prodrug, the active form being its intracellular AppCCl2p metabolite.

Adenosine Triphosphate↗
PubMed2001

Structure-activity relationships for inhibition of farnesyl diphosphate synthase in vitro and inhibition of bone resorption in vivo by nitrogen-containing bisphosphonates.

It has long been known that small changes to the structure of the R(2) side chain of nitrogen-containing bisphosphonates can dramatically affect their potency for inhibiting bone resorption in vitro and in vivo, although the reason for these differences in antiresorptive potency have not been explained at the level of a pharmacological target. Recently, several nitrogen-containing bisphosphonates were found to inhibit osteoclast-mediated bone resorption in vitro by inhibiting farnesyl diphosphate synthase, thereby preventing protein prenylation in osteoclasts. In this study, we examined the potency of a wider range of nitrogen-containing bisphosphonates, including the highly potent, heterocycle-containing zoledronic acid and minodronate (YM-529). We found a clear correlation between the ability to inhibit farnesyl diphosphate synthase in vitro, to inhibit protein prenylation in cell-free extracts and in purified osteoclasts in vitro, and to inhibit bone resorption in vivo. The activity of recombinant human farnesyl diphosphate synthase was inhibited at concentrations > or = 1 nM zoledronic acid or minodronate, the order of potency (zoledronic acid approximately equal to minodronate > risedronate > ibandronate > incadronate > alendronate > pamidronate) closely matching the order of antiresorptive potency. Furthermore, minor changes to the structure of the R(2) side chain of heterocycle-containing bisphosphonates, giving rise to less potent inhibitors of bone resorption in vivo, also caused a reduction in potency up to approximately 300-fold for inhibition of farnesyl diphosphate synthase in vitro. These data indicate that farnesyl diphosphate synthase is the major pharmacological target of these drugs in vivo, and that small changes to the structure of the R(2) side chain alter antiresorptive potency by affecting the ability to inhibit farnesyl diphosphate synthase.

Alkyl and Aryl Transferases↗
PubMed2000

Helicobacter pylori-selective antibacterials based on inhibition of pyrimidine biosynthesis.

We report the discovery of a class of pyrazole-based compounds that are potent inhibitors of the dihydroorotate dehydrogenase of Helicobacter pylori but that do not inhibit the cognate enzymes from Gram-positive bacteria or humans. In culture these compounds inhibit the growth of H. pylori selectively, showing no effect on other Gram-negative or Gram-positive bacteria or human cell lines. These compounds represent the first examples of H. pylori-specific antibacterial agents. Cellular activity within this structural class appears to be due to dihydroorotate dehydrogenase inhibition. Minor structural changes that abrogate in vitro inhibition of the enzyme likewise eliminate cellular activity. Furthermore, the minimum inhibitory concentrations of these compounds increase upon addition of orotate to the culture medium in a concentration-dependent manner, consistent with dihydroorotate dehydrogenase inhibition as the mechanism of cellular inhibition. The data presented here suggest that targeted inhibition of de novo pyrimidine biosynthesis may be a valuable mechanism for the development of antimicrobial agents selective for H. pylori.

Amino Acid Sequence↗
PubMed2000

Selective inhibition of bacterial dihydroorotate dehydrogenases by thiadiazolidinediones.

Dihydroorotate dehydrogenase is a critical enzyme of de novo pyrimidine biosynthesis in prokaryotic and eukaryotic cells. Differences in the primary structure of the enzymes from Gram-positive and -negative bacteria and from mammals indicate significant structural divergence among these enzymes. We have identified a class of small molecules, the thiadiazolidinediones, that inhibit prototypical enzymes from Gram-positive and -negative bacteria, but are inactive against the human enzyme. The most potent compound in our collection functioned as a time-dependent irreversible inactivator of the bacterial enzymes with k(inact)/K(i) values of 48 and 500 M(-1) sec(-1) for the enzymes from Escherichia coli and Enterococcus faecalis, respectively. The data presented here indicate that it is possible to inhibit prokaryotic dihydroorotate dehydrogenases selectively while sparing the mammalian enzyme. Thus, this enzyme may represent a valuable target for the development of novel antibiotic compounds.

Anti-Bacterial Agents↗
PubMed2000

Cellular and molecular mechanisms of action of bisphosphonates.

BACKGROUND: Bisphosphonates currently are the most important class of antiresorptive agents used in the treatment of metabolic bone diseases, including tumor-associated osteolysis and hypercalcemia, Paget's disease, and osteoporosis. These compounds have high affinity for calcium and therefore target to bone mineral, where they appear to be internalized selectively by bone-resorbing osteoclasts and inhibit osteoclast function. METHODS: This article reviews the pharmacology of bisphosphonates and the relation between the chemical structure of bisphosphonates and antiresorptive potency, and describes recent new discoveries of their molecular mechanisms of action in osteoclasts. RESULTS: Bisphosphonates can be grouped into two pharmacologic classes with distinct molecular mechanisms of action. Nitrogen-containing bisphosphonates (the most potent class) act by inhibiting the mevalonate pathway in osteoclasts, thereby preventing prenylation of small GTPase signaling proteins required for osteoclast function. Bisphosphonates that lack a nitrogen in the chemical structure do not inhibit protein prenylation and have a different mode of action that may involve the formation of cytotoxic metabolites in osteoclasts or inhibition of protein tyrosine phosphatases. CONCLUSIONS: Bisphosphonates are highly effective inhibitors of bone resorption that selectively affect osteoclasts. After more than 30 years of clinical use, their molecular mechanisms of action are only just becoming clear.

Animals↗
PubMed2000

A second dihydroorotate dehydrogenase (Type A) of the human pathogen Enterococcus faecalis: expression, purification, and steady-state kinetic mechanism.

We report the identification, expression, and characterization of a second Dihydroorotate dehydrogenase (DHODase A) from the human pathogen Enterococcus faecalis. The enzyme consists of a polypeptide chain of 322 amino acids that shares 68% identity with the cognate type A enzyme from the bacterium Lactococcus lactis. E. faecalis DHODase A catalyzed the oxidation of l-dihydroorotate while reducing a number of substrates, including fumarate, coenzyme Q(0), and menadione. The steady-state kinetic mechanism has been determined with menadione as an oxidizing substrate at pH 7.5. Initial velocity and product inhibition data suggest that the enzyme follows a two-site nonclassical ping-pong kinetic mechanism. The absorbance of the active site FMN cofactor is quenched in a concentration-dependent manner by titration with orotate and barbituric acid, two competitive inhibitors with respect to dihydroorotate. In contrast, titration of the enzyme with menadione had no effect on FMN absorbance, consistent with nonoverlapping binding sites for dihyroorotate and menadione, as suggested from the kinetic mechanism. The reductive half-reaction has been shown to be only partially rate limiting, and an attempt to evaluate the slow step in the overall reaction has been made by simulating orotate production under steady-state conditions. Our data indicate that the oxidative half-reaction is a rate-limiting segment, while orotate, most likely, retains significant affinity for the reduced enzyme, as suggested by the product inhibition pattern.

Amino Acid Sequence↗
PubMed2000

Analysis of an adenine nucleotide-containing metabolite of clodronate using ion pair high-performance liquid chromatography-electrospray ionisation mass spectrometry.

Clodronate belongs to the family of bisphosphonates, which are synthetic analogues of pyrophosphate. Bisphosphonates are widely used in the treatment of metabolic bone diseases. Some bisphosphonates, including clodronate, can be metabolized in cells into non-hydrolysable nucleotide analogues. In this paper, we describe a new method for extraction and quantitation of the clodronate metabolite in cell lysates by using ion-pairing HPLC method that is compatible with negative ion electrospray ionization mass spectrometry (ESI-MS). The method was used for detection of the metabolite of clodronate in extracts from RAW 264 macrophage cells after treatment with clodronate.

Adenine Nucleotides↗
PubMed2000

A deletion on chromosome 4 cosegregates with the whirler deafness mutation: exclusion of Orm1 as a candidate.

Whirler (wi) mice display profound deafness and a head-tossing and circling phenotype, showing an autosomal recessive mode of inheritance. The wi mutation has been shown to map close to the Orm gene cluster on mouse Chromosome (Chr) 4. We have, therefore, investigated the Orm loci as candidates for the whirler gene. Detailed mapping and analysis of the Orm gene cluster in both normal and whirler mice indicates the presence of a <48-kb deletion in whirler mice that disrupts the Orm1 locus. The Orm1 locus is also deleted in the CE/J mouse strain, and we discuss the candidature of Orm1 for the whirler gene.

Animals↗
PubMed2000

Effects of interruption of apicoplast function on malaria infection, development, and transmission.

A chloroplast-like organelle is present in many species of the Apicomplexa phylum. We have previously demonstrated that the plastid organelle of Plasmodium faciparum is essential to the survival of the blood-stage malaria parasite in culture. One known function of the plastid organelle in another Apicomplexan, Toxoplasma gondii, involves the formation of the parasitophorous vacuole. The effects of interruption of plastid function on sporozoites and sexual-stage parasites have not been investigated. In our previous studies of the effects of thiostrepton, a polypeptide antibiotic from streptococcus spp., on erythrocytic schizongony of the human malaria P. falciparium, we found that this antibiotic appears to interact with the guanosine triphosphatase (GTPase) binding domain of the organellar large subunit ribosomal RNA, as it does in bacteria. We investigate here the effects of this drug on life-cycle stages of the malaria parasite in vivo. Preincubation of mature infective sporozoites with thiostrepton has no observable effect on their infectivity. Sporozoite infection both by mosquito bite and sporozoite injection was prevented by pretreatment of mice with thiostrepton. Thiostrepton eliminates infection with erythrocytic forms of Plasmodium berghei in mice. Clearance of infected red blood cells follows the delayed kinetics associated with drugs that interact with the apicoplast. Thiostrepton treatment of infected mice reduces transmission of parasites by more than ten-fold, indicating that the plastid has a role in sexual development of the parasite. These results indicate that the plastid function is accessible to drug action in vivo and important to the development of both sexual and asexual forms of the parasite.

Animals↗
PubMed2000

The potent bisphosphonate ibandronate does not induce myeloma cell apoptosis in a murine model of established multiple myeloma.

Bisphosphonates are effective in the management of bone disease in patients with multiple myeloma and recent reports have suggested that they may also have an anti-tumour activity. In support of this, we have previously demonstrated that bisphosphonates can induce myeloma cell apoptosis in vitro; however, it remains unclear whether this occurs in vivo. We have therefore investigated the effect of the potent bisphosphonate ibandronate in the 5T2MM murine model of established multiple myeloma. Short-term treatment with a high dose of ibandronate had no effect on either myeloma cell number or the proportion of myeloma cells undergoing apoptosis. These observations suggest that although bisphosphonates induce apoptosis in myeloma cells in vitro, they may not have the same anti-tumour effects in vivo.

Animals↗