ORCID Profile
0000-0002-9932-4042
Current Organisation
University of Oxford
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Publisher: Elsevier BV
Date: 09-2014
DOI: 10.1016/J.EJMECH.2014.06.062
Abstract: Phosphonocarboxylate (PC) analogs of the anti-osteoporotic drugs, bisphosphonates, represent the first class of selective inhibitors of Rab geranylgeranyl transferase (RabGGTase, RGGT), an enzyme implicated in several diseases including ovarian, breast and skin cancer. Here we present the synthesis and biological characterization of an extended set of this class of compounds, including lipophilic derivatives of the known RGGT inhibitors. From this new panel of PCs, we have identified an inhibitor of RGGT that is of similar potency as the most active published phosphonocarboxylate, but of higher selectivity towards this enzyme compared to prenyl pyrophosphate synthases. New insights into structural requirements are also presented, showing that only PC analogs of the most potent 3rd generation bisphosphonates inhibit RGGT. In addition, the first phosphonocarboxylate-derived GGPPS inhibitor is reported.
Publisher: Elsevier BV
Date: 05-2005
Publisher: American Chemical Society (ACS)
Date: 08-03-2008
DOI: 10.1021/JM7015733
Abstract: The nitrogen-containing bisphosphonates (N-BPs) are the main drugs currently used to treat diseases characterized by excessive bone resorption. The major molecular target of N-BPs is farnesylpyrophosphate synthase. N-BPs inhibit the enzyme by a mechanism that involves time dependent isomerization of the enzyme. We investigated features of N-BPs that confer maximal slow and tight-binding by quantifying the initial and final K(i)s and calculating the isomerization constant K(isom) for many N-BPs. Disruption of the phosphonate-carbon-phosphonate backbone resulted in loss of potency and reduced K(isom). The lack of a hydroxyl group on the geminal carbon also reduced K(isom). The position of the nitrogen in the side chain was crucial to both K(i) and K(isom). A correlation of K(isom) and also final K(i) with previously published in vivo potency reveals that the isomerization constant ( R = -0.77, p < 0.0001) and the final inhibition of FPPS by N-BPs ( R = 0.74, p < 0.0001) are closely linked to antiresorptive efficacy.
Publisher: American Chemical Society (ACS)
Date: 15-04-2010
DOI: 10.1021/JM900232U
Abstract: 3-(3-Pyridyl)-2-hydroxy-2-phosphonopropanoic acid (3-PEHPC, 1) is a phosphonocarboxylate (PC) analogue of 2-(3-pyridyl)-1-hydroxyethylidenebis(phosphonic acid) (risedronic acid, 2), an osteoporosis drug that decreases bone resorption by inhibiting farnesyl pyrophosphate synthase (FPPS) in osteoclasts, preventing protein prenylation. 1 has lower bone affinity than 2 and weakly inhibits Rab geranylgeranyl transferase (RGGT), selectively preventing prenylation of Rab GTPases. We report here the synthesis and biological studies of 2-hydroxy-3-imidazo[1,2-a]pyridin-3-yl-2-phosphonopropionic acid (3-IPEHPC, 3), the PC analogue of minodronic acid 4. Like 1, 3 selectively inhibited Rab11 vs. Rap 1A prenylation in J774 cells, and decreased cell viability, but was 33-60x more active in these assays. After resolving 3 by chiral HPLC (>98% ee), we found that (+)-3-E1 was much more potent than (-)-3-E2 in an isolated RGGT inhibition assay, approximately 17x more potent (LED 3 microM) than (-)-3-E2 in inhibiting Rab prenylation in J774 cells and >26x more active in the cell viability assay. The enantiomers of 1 exhibited a 4-fold or smaller potency difference in the RGGT and prenylation inhibition assays.
Publisher: Wiley
Date: 11-2007
Abstract: The bisphosphonates (BPs) are well established as the treatments of choice for disorders of excessive bone resorption, including Paget's disease of bone, myeloma and bone metastases, and osteoporosis. There is considerable new knowledge about how BPs work. Their classical pharmacological effects appear to result from two key properties: their affinity for bone mineral and their inhibitory effects on osteoclasts. Mineral binding affinities differ among the clinically used BPs and may influence their differential distribution within bone, their biological potency, and their duration of action. The inhibitory effects of the nitrogen-containing BPs (including alendronate, risedronate, ibandronate, and zoledronate) on osteoclasts appear to result from their inhibition of farnesyl pyrophosphate synthase (FPPS), a key branch-point enzyme in the mevalonate pathway. FPPS generates isoprenoid lipids used for the posttranslational modification of small GTP-binding proteins essential for osteoclast function. Effects on other cellular pathways, such as preventing apoptosis in osteocytes, are emerging as other potentially important mechanisms of action. As a class, BPs share several common properties. However, as with other classes of drugs, there are obvious chemical, biochemical, and pharmacological differences among the various in idual BPs. Each BP has a unique profile that may help to explain potential important clinical differences among the BPs, in terms of speed of onset of fracture reduction, antifracture efficacy at different skeletal sites, and the degree and duration of suppression of bone turnover. As we approach the 40th anniversary of the discovery of their biological effects, there remain further opportunities for using their properties for medical purposes.
Publisher: Elsevier BV
Date: 2008
DOI: 10.1016/J.BBRC.2007.11.054
Abstract: Chemically modified tetracyclines (CMTs 1-10) were developed as non-antibiotic inhibitors of matrix metalloproteinases (MMPs). We previously demonstrated that MMP inhibition alone is insufficient to explain the pro-apoptotic action of CMTs in osteoclast lineage cells and we have explored additional mechanisms of action. We compared the characteristics of apoptosis in RAW264.7 murine monocyte and osteoclast cultures treated with pharmacologically relevant concentrations of CMT3 or the bisphosphonate alendronate, which induces osteoclast apoptosis through inhibition of farnesyl diphosphate synthase. CMT3 induced apoptosis rapidly (2-3h), whereas alendronate-induced apoptosis was delayed (>12h). CMT3-treated cells did not accumulate unprenylated Rap1A in contrast to cells treated with alendronate. Importantly, CMT3 induced a rapid loss of mitochondrial stability in RAW264.7 cells measured by loss of Mitotracker((R)) Red fluorescence, while bongkrekic acid protected polykaryons from CMT3-induced apoptosis. Modulation of mitochondrial function is therefore a significant early action of CMT3 that promotes apoptosis in osteoclast lineage cells.
Publisher: American Chemical Society (ACS)
Date: 11-07-2012
DOI: 10.1021/JM300677J
Publisher: Elsevier BV
Date: 2002
Publisher: Proceedings of the National Academy of Sciences
Date: 16-05-2006
Abstract: Osteoporosis and low bone mass are currently estimated to be a major public health risk affecting % 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.
Publisher: Wiley
Date: 05-2006
DOI: 10.1359/JBMR.060118
Abstract: 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. Nitrogen-containing bisphosphonates (N-BPs) are potent inhibitors of bone resorption that act by inhibiting farnesyl diphosphate synthase, thereby indirectly preventing the prenylation of Rho family GTPases that are required for the function and survival of bone-resorbing osteoclasts. However, the effect that these drugs have on the activity of Rho family GTPases has not been determined. The effect of N-BPs on the activity of Rho family GTPases in J774 macrophages and osteoclasts was measured using a pull-down assay to isolate the GTP-bound forms. The effect of N-BPs, or decreasing Rac expression using siRNA, on downstream p38 activity was evaluated by Western blotting and apoptosis assessed by measurement of caspase 3/7 activity. Rather than inhibiting GTPase function, loss of prenylation after treatment with N-BPs caused an increase in the GTP-bound form of Rac, Cdc42, and Rho in J774 cells and osteoclast-like cells, which paralleled the rate of accumulation of unprenylated small GTPases. Activation of Rac also occurred with other inhibitors of prenylation of Rho-family proteins, such as mevastatin and the geranylgeranyl transferase I inhibitor GGTI-298. The Rac-GTP that increased after N-BP treatment was newly translated, cytoplasmic unprenylated protein, because it was not labeled with [(14)C] mevalonate, and the increase in Rac-GTP was prevented by cycloheximide. Furthermore, this unprenylated Rac-GTP retained at least part of its functional activity in J774 cells, because it mediated N-BP-induced activation of p38. Paradoxically, although risedronate induces apoptosis of J774 macrophages by inhibiting protein prenylation, the p38 inhibitor SB203580 enhanced N-BP-induced apoptosis, suggesting that Rac-induced p38 activation partially suppresses the pro-apoptotic effect of N-BPs in these cells. N-BP drugs may disrupt the function of osteoclasts in vivo and affect other cell types in vitro by inhibiting protein prenylation, thereby causing inappropriate and sustained activation, rather than inhibition, of some small GTPases and their downstream signaling pathways.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 09-2007
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for James Dunford.