Pharmacology of bisphosphonates Dr. Fraser Coxon Bone & Musculoskeletal Research Programme Institute of Medical ciences University of Aberdeen
utline Introduction to osteoclasts Culture and analysis of osteoclasts in vitro Mechanism of action of bisphosphonates Mechanism of action of phosphonocarboxylate analogues of bisphosphonates Localisation of these compounds in bone
Formation of osteoclasts steoclast precursor NFκB M-CF osteoblast 1,25() 2 D 3 PTH estrogen Pre- steoclast c-fos RANKL RANK Mature steoclast RANK = receptor activator of NFκB RANKL = RANK ligand
Identifying features of osteoclasts Multinucleated Have high levels of TRAP (tartrate( tartrate-resistant resistant acid phosphatase) Abundance of acidic vesicles α v β 3 integrin (vitronectin receptor) on the cell surface Expression of cathepsinc K Phase contrast taining for TRAP activity vitronectin receptor (green) acidic vesicles (red) Cath K activity (red) (live osteoclast)
Identifying features of resorbing osteoclasts Formation of distinct membrane domains (e.g. ruffled border) Polarisation of cytoskeleton into an F-actin F ring Resorption of bone! Actin rings visualised by staining with fluorescent phalloidin conjugates Resorption pits on bone surface visualised reflected light microscopy
steoclasts are specialised bone-resorbing cells F-actin ring sealing zone H + CK H + CK H + BNE ruffled border Bone is resorbed by the secretion of acid and cathepsin K (CK) at the ruffled border of the osteoclast
Analysis of resorbing osteoclasts steoclasts need to be cultured on a mineralised substrate Dentine Cortical bone Hydroxyapatite discs axial views: hsteoclasts on dentine 30µm hsteoclast on glass 10µm
Unusual membrane domains in osteoclasts basolateral apical basolateral apical Vesicular stomatitis virus Influenza virus - basolateral domain in epithelial cells - apical domain in epithelial cells alo et al. 1996 J Cell ci 109,, 391 LAMP-2 Ruffled border: Properties of an endosomal/lysosomal membrane Vitronectin receptor Palokangas et al 1997 J Cell ci 110,, 1767
Unique membrane domains in osteoclasts FD (apical domain) FD (apical domain) basolateral domain BL BL basolateral domain Z Z sealing zone ruffled border Bone FD- Functional secretory domain Z- sealing zone (F-actin ring)
Vesicular trafficking routes in osteoclasts FD (apical domain) FD (apical domain) basolateral domain BL BL basolateral domain Z Z sealing zone ruffled border Bone FD- Functional secretory domain Z- sealing zone (F-actin ring)
ources of osteoclast cultures
Isolation of mature osteoclasts Mince and scrape the bones to release osteoclasts washing or digestion relatively low numbers impure cultures useful for resorption assays or single cell studies Mixed culture of osteoclasts/other bone cells Pure culture of TRAP +ve osteoclasts
Generation of osteoclasts in vitro flush bone marrow from long bones high numbers fairly pure cultures useful for formation assays or biochemical analyses Isolate PBMCs from human blood eed cells in petri-dishes M-CF M-CF M-CF + RANKL M-CF + RANKL M-CF + RANKL Day 0 Day 3 Day 6 Re-seed on to resorbable substrate e.g. dentine cortical bone Day 9 Day 12 Day 15 Resorbing osteoclasts
Generation of osteoclasts in vitro- before RANKL! flush bone marrow from long bones Isolate osteoblasts from calvaria steoclast Co-culture cells in the presence of 1,25()2D3 ~ 8 Days Useful for assessing osteoblast-osteoclast interactions in osteoclast formation e.g. whether a defect is in the osteoblast or osteoclast lineage
Isolation of osteoclasts ex vivo Isolate osteoclasts using antibody to surface marker (e.g. VNR) and magnetic bead separation Pure culture of VNR +ve osteoclasts Useful for assessing the effect of a drug on osteoclasts in vivo
Bisphosphonates
Bisphosphonates bind calcium ions and target bone mineral ide chain R 1 C = P P = - - Ca 2+
Localisation of a fluorescent BP (RI) in mouse vertebra cartilage Bone marrow trabecular bone Bone Green = FAM-RI Blue = nuclei 0.1 mm
steoclasts engulf bisphosphonate into intracellular vesicles Coxon et al 2008, Bone 42:848-60 osteoclast resorption pit dentine surface zx Green- F-ALN Red- F-actin Blue-VNR green = bisphosphonate red = actin cytoskeleton
Bisphosphonates are selectively internalised from mineralised surfaces by osteoclasts Rabbit osteoclasts cultured on dentine labelled with fluorescent alendronate Bisphosphonate * * * * dextran (endocytic marker) * * * Green- F-ALN Red- F-actin Blue-VNR Green- dextran Red- F-actin Blue-VNR
Effects of BPs on osteoclast morphology cytoskeleton (F-actin ring) Ruffled border in vivo cell morphology in vitro resorbing osteoclast retraction apoptosis + RI ato et al 1991, J Clin Invest 88, 2095
Two groups of of bisphosphonates with different modes of of action imple Bisphosphonates Nitrogen-containing Bisphosphonates H 3 C Cl Cl H = P P = clodronate = P P = etidronate H 2 N N H pamidronate N = P P = H zoledronate = P P = CH 3 N H = P P = H 2 N N H ibandronate = P P = alendronate H = P P = risedronate
Bisphosphonates inhibit farnesyl diphosphate synthase in the mevalonate pathway Luckman et al,, 1998 Fisher et al,, 1999 Van Beek et al,, 1999 Keller et al,, 1999 Bergstrom et al,, 2000 Grove et al,, 2000 Dunford et al,, 2001 HMG-CoA mevalonate phosphomevalonate mevalonate diphosphate cholesterol dimethylallyl diphosphate squalene x geranyl diphosphate farnesyl diphosphate isopentenyl diphosphate FPP synthase N-BPs e.g. pamidronate alendronate Ibandronate risedronate zoledronate geranylgeranyl diphosphate
Most small GTPases are modified by prenylation -P - - P farnesyl diphosphate farnesyl transferase Ras CAA X Ras proteins Rab CXC Rab proteins -P - - P Rab geranylgeranyl transferase geranylgeranyl diphosphate geranylgeranyl Transferase I Rho CAA X Rho proteins Rap1
mall GTPases act as as molecular switches Guanine nucleotide exchange factor (GEF) GDP Ras ff GTP GEF GDP n GDI GDP Rho GDP REP Rab Translocation to membrane GTPase GDP Pi GAP GTPase activating protein (GAP) GTPase GTP Effector downstream signalling
Bisphosphonates inhibit protein prenylation Ctl HMGCoA A B A- Rab GTPases B- 21 kda GTPases [ 14 C]mevalonate dimethylallyl diphosphate isopentenyl diphosphate geranyl diphosphate FPP synthase farnesyl diphosphate FTase Ras 14 C] Ras proteins [ 14 Ras Rab [ 14 14 C] Rab proteins Geranylgeranyl Rab GGTase diphosphate GGTase I Rho Rho [ 14 family C] Rho Rap1 proteins Rap1
Bisphosphonates inhibit protein prenylation Ctl RI HMGCoA A B A- Rab GTPases B- 21 kda GTPases [ 14 C]mevalonate dimethylallyl diphosphate isopentenyl diphosphate BPs e.g. RI FPP synthase FTase Ras Rab GGTase GGTase I Rab Rho
RI disrupts the localisation of Rab6 in rabbit osteoclasts Ctrl Rab6 WGA (Golgi) merge RI
Loss of prenylated small GTPases disrupts osteoclast function cytoskeleton disruption Induction of apoptosis loss of prenylated small GTPases Rho, Rac, Cdc42 (regulate the actin cytoskeleton) Rho, Rac (regulate cell survival) Loss of the ruffled border Rabs (regulate vesicular Transport)
FPP synthase catalyses the condensation of of isoprenoid lipid chains 5-carbon 5-carbon IPP + DMAPP 10-carbon GPP + IPP 15-carbon FPP = P - P - = P - P - = P - P - = P - P - = = = = DMAPP GPP substrate DMAPP/GPP FPP synthase IPP substrate
Risedronate Risedronateco-ordinates co-ordinateswith with33magnesium magnesiumions ionsin in the the GGP GGPbinding bindingsite siteof offpp FPP Kavanagh et al 2006, Proc. Natl. Acad. ci 103, 7829-7834
Courtesy of Jean-Michel Rondeau,, Novartis
Correlation between inhibition of of FPP synthase in in vitro and anti-resorptive potency in in vivo Dunford et al, J Pharm Exp Ther 2001 10000 IC 50 (nm) for inhibition of rhfpp synthase 1000 r=0.95, p<0.0001 100 10 1 0.0001 0.001 0.01 0.1 1 Lowest effective dose (mgp( mgp/kg) in vivo
The nitrogen in in the side chain of of risedronate or or zoledronate interacts with a conserved threonine and lysine residue in in FPP synthase ideal distance is ~3Å Zoledronate Risedronate Alendronate Ibandronate 2.96 2.97 3.84 4.03 Risedronate Kavanagh et al 2006 Proc. Natl. Acad. ci Zoledronate Rondeau JM et al 2006 Chem Med Chem This interaction enables slow tight binding of the enzyme
Phosphonocarboxylate analogue of RI Bisphosphonate Phosphonocarboxylate N H P P Risedronate (RI) N H P C NE10790 (3-PEHPC) 50x lower affinity for bone 5,000x lower anti-resorptive potency
NE10790 is a novel inhibitor of Rab GGTase Ctl RI [ 14 C]mevalonate Coxon et al 2001 J Biol Chem 276,, 48213 A B DMAPP/IPP A- Rab GTPases B- 21 kda GTPases FPP synthase farnesyl diphosphate (FPP) FTase [ 14 Ras C] Ras 14 C] Ras [ 14 14 C] Rab proteins Geranylgeranyl diphosphate (GGPP) Rab GGTase GGTase I [ 14 C] Rho proteins Rap1 Rab Rho Rho Rab
NE10790 is a novel inhibitor of Rab GGTase Ctl RI NE10790 [ 14 C]mevalonate Coxon et al 2001 J Biol Chem 276,, 48213 A B DMAPP/IPP A- Rab GTPases B- 21 kda GTPases FPP synthase NE10790 farnesyl diphosphate (FPP) FTase [ 14 Ras C] Ras 14 C] Rab Rab Rab Geranylgeranyl diphosphate (GGPP) Rab GGTase GGTase I Ras [ 14 C] Rho proteins Rap1 Rho Rho
NE10790 disrupts localisation of Rab6 in rabbit osteoclasts Ctrl Rab6 WGA (Golgi) merge NE10790
Family of small GTPases (21-28kDa) 28kDa) with more than 70 human isoforms The Rab family of small GTPases Rab GTPases are crucial regulators of vesicle trafficking: Vesicle formation Vesicle motility Vesicle docking Membrane fusion Try chwartz? chwartz et al (2007) J Cell ci 120: 3905-3910 3910
% of ctrl NE10790 inhibits bone resorption in vitro without affecting the cytoskeleton 140 120 100 80 60 40 20 0 RI RI 0.1 1 10 100 Concentration (µm)( % of ctrl 140 120 100 80 60 40 20 0 NE10790 NE10790 0.1 0.5 1 Concentration (mm( mm) Resorption area actin rings
Inhibition of Rab prenylation alters vesicular trafficking in osteoclasts in vitro Control Coxon et al 2001, J Biol Chem 276, 48213-48222 48222 1mM NE10790
EM Inhibition of Rab prenylation alters vesicular trafficking in osteoclasts in vitro Coxon et al 2001, J Biol Chem 276, 48213-48222 48222 Control 1mM NE10790 confocal (axial views) *
Do these drugs inhibit protein prenylation in osteoclasts in vivo?
Do Do these compounds inhibit protein prenylation in in vivo? 2mgP/kg NE10790 or RI 24 hrs 23c6 & anti- mouse IgG- coated Dynal beads VNR +ve osteoclasts Purified osteoclast pellet In vitro prenylation assay using rh Rab GGTase Assay for unprenylated Rap1A
BPs and PCs inhibit protein prenylation in osteoclasts in vivo Unprenylated Rabs detected by prenylating with [ 3 H]GGPP in vitro: +ve: : osteoclast fraction -ve:: non-osteoclast osteoclast bone marrow cells J774 cells in vitro Ctl NE10790 CTL +ve -ve 0.5mg/kg RI +ve -ve 45mg/kg PC +ve -ve Rab Rab Rab proteins prenylated in vitro Rab Rab Rab in vitro prenylation assay western blotting for unprenylated Rap1A: CTL RI PC Rab Rab Rab Rab Rab 3 H 3 H unprenylated Rap1A actin +ve -ve +ve -ve +ve -ve
Are the effects of BPs really due to inhibition of FPP?
Loss of geranylgeranylated proteins accounts for inhibition of resorption by BPs Fisher et al 1999, Proc Natl Acad ci, 96:133-138; Coxon et al 2000, J Bone Miner Res 15:1467-1476 1476 120 mevalonate 100 ALN Bone resorption (% of control) 80 60 40 Farnesol (F) farnesyl diphosphate farnesylated proteins 20 0 Ctrl 60µM ALN ALN+ GG ALN+ F geranylgeranyl diphosphate Geranylgeranylated Rho proteins Risedronate (µm) + GG 0 12.5 25 50 12.5 25 50 β-actin geranylgeraniol (GG) Geranylgeranylated Rab proteins unprenylated Rab 6
Loss of geranylgeranylated proteins accounts for inhibition of resorption by BPs Coxon et al 2000, J Bone Miner Res 15:1467-1476 1476 Coxon et al 2003, Calcif Tissue Int 72:80-84 84 mevalonate steoclast polarisation: BPs farnesyl diphosphate FTI-277 farnesylated proteins Ctrl steoclast resorption: Resorption GGTI-298 area (mm 2 ) 0.1 geranylgeranyl 0.08 Geranylgeranylated diphosphate Rho proteins 0.06 0.04 0.02 Geranylgeranylated 0 Rab proteins Ctrl 10µM FTI 5µM10µM FTI FTI 10µM GGTI 5µM 10µM GGTI GGTI
ummary Assays using osteoclasts in vitro and ex vivo have enabled us to determine the mechanism of action of bisphosphonates and the related phosphonocarboxylates Inhibition of prenylation of small GTPases Morphological analysis of osteoclasts treated with these compounds has also shed light on the importance of prenylated small GTPases for osteoclast function
Localisation of BPs and PCs in bone
Bisphosphonates have different affinities for hydroxyapatite Nancollas et al 2006, Bone 38: 617-627 627 The R 2 side-chain of BPs also contributes to bone affinity HAP Adsorption Affinity Constants at ph 7.4 4 KL/10 6 L mol -1 3 2 1 ZL ALN RI 0 CL ETD RI IBN ALN ZL Lawson, Triffitt, Ebetino & Russell ABMR & Davos 2005 & 2006
Are osteocytes exposed to bisphosphonates in vivo? Photo by Lilian Plotkin and Lynda Bonewald Lower affinity BPs should be able to gain access to more sites in bone than higher affinity BPs which will get stuck at sites of first contact
Anti-apoptotic effect of BPs on osteocytes (ML-Y4 cells) BPs can open Connexin 43 hemichannels and activate anti-apoptotic apoptotic signalling pathways via ERK % etoposide- induced apoptosis 150 100 50 0 risedronate * * * * 150 100 50 0 etidronate * * * * * * -50 0 10-11 10-10 10-9 10-8 10-7 10-6 10-5 -50 0 10-11 10-10 10-9 10-8 10-7 10-6 10-5 concentration (M) concentration (M) IC 50 values in the nm range! Plotkin, Manolagas, Bellido, Bone 39 (2006) 443-452
Methods Mice or rats injected subcutaneously with fluorescently labelled conjugates (fluorescein, rhodamine or AlexaFluor 647) of RI and 3-PEHPC Animals sacrificed 24h later and tibiae fixed and embedded in MMA resin Resin blocks cut transversely and bone examined directly by confocal microscopy
F-RI localises to osteocyte lacunae in mouse cortical bone osteocyte Vascular channel Green- F-RI Blue-nuclei Red- wheat germ agglutinin
Acknowledgements Bone Research Group, Aberdeen Mike Rogers Miep Helfrich Adam Taylor Anke Roelofs Debbie cott Aysha Khalid University of xford Graham Russell Jim Dunford Procter & Gamble Pharmaceuticals Hal Ebetino Mark Lundy University of outhern California Charles McKenna Imperial College, London Miguel eabra Mimi Mules Queen Mary, University of London Alan Boyde