Pharmacology of bisphosphonates

Transcription

1 Pharmacology of bisphosphonates Dr. Fraser Coxon Bone & Musculoskeletal Research Programme Institute of Medical ciences University of Aberdeen

2 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

3 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

4 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)

5 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

6 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

7 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

8 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 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

9 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)

10 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)

11 ources of osteoclast cultures

12 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

13 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

14 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

15 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

16 Bisphosphonates

17 Bisphosphonates bind calcium ions and target bone mineral ide chain R 1 C = P P = - - Ca 2+

18 Localisation of a fluorescent BP (RI) in mouse vertebra cartilage Bone marrow trabecular bone Bone Green = FAM-RI Blue = nuclei 0.1 mm

19 steoclasts engulf bisphosphonate into intracellular vesicles Coxon et al 2008, Bone 42: osteoclast resorption pit dentine surface zx Green- F-ALN Red- F-actin Blue-VNR green = bisphosphonate red = actin cytoskeleton

20 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

21 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

22 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

23 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

24 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

25 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

26 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 [ C] Rab proteins Geranylgeranyl Rab GGTase diphosphate GGTase I Rho Rho [ 14 family C] Rho Rap1 proteins Rap1

27 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

28 RI disrupts the localisation of Rab6 in rabbit osteoclasts Ctrl Rab6 WGA (Golgi) merge RI

29 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)

30 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

31 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,

32 Courtesy of Jean-Michel Rondeau,, Novartis

33 Correlation between inhibition of of FPP synthase in in vitro and anti-resorptive potency in in vivo Dunford et al, J Pharm Exp Ther IC 50 (nm) for inhibition of rhfpp synthase 1000 r=0.95, p< Lowest effective dose (mgp( mgp/kg) in vivo

34 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 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

35 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

36 NE10790 is a novel inhibitor of Rab GGTase Ctl RI [ 14 C]mevalonate Coxon et al 2001 J Biol Chem 276,, A B DMAPP/IPP A- Rab GTPases B- 21 kda GTPases FPP synthase farnesyl diphosphate (FPP) FTase [ 14 Ras C] Ras 14 C] Ras [ C] Rab proteins Geranylgeranyl diphosphate (GGPP) Rab GGTase GGTase I [ 14 C] Rho proteins Rap1 Rab Rho Rho Rab

37 NE10790 is a novel inhibitor of Rab GGTase Ctl RI NE10790 [ 14 C]mevalonate Coxon et al 2001 J Biol Chem 276,, 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

38 NE10790 disrupts localisation of Rab6 in rabbit osteoclasts Ctrl Rab6 WGA (Golgi) merge NE10790

39 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:

40 % of ctrl NE10790 inhibits bone resorption in vitro without affecting the cytoskeleton RI RI Concentration (µm)( % of ctrl NE10790 NE Concentration (mm( mm) Resorption area actin rings

41 Inhibition of Rab prenylation alters vesicular trafficking in osteoclasts in vitro Control Coxon et al 2001, J Biol Chem 276, mM NE10790

42 EM Inhibition of Rab prenylation alters vesicular trafficking in osteoclasts in vitro Coxon et al 2001, J Biol Chem 276, Control 1mM NE10790 confocal (axial views) *

43 Do these drugs inhibit protein prenylation in osteoclasts in vivo?

44 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

45 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

46 Are the effects of BPs really due to inhibition of FPP?

47 Loss of geranylgeranylated proteins accounts for inhibition of resorption by BPs Fisher et al 1999, Proc Natl Acad ci, 96: ; Coxon et al 2000, J Bone Miner Res 15: mevalonate 100 ALN Bone resorption (% of control) Farnesol (F) farnesyl diphosphate farnesylated proteins 20 0 Ctrl 60µM ALN ALN+ GG ALN+ F geranylgeranyl diphosphate Geranylgeranylated Rho proteins Risedronate (µm) + GG β-actin geranylgeraniol (GG) Geranylgeranylated Rab proteins unprenylated Rab 6

48 Loss of geranylgeranylated proteins accounts for inhibition of resorption by BPs Coxon et al 2000, J Bone Miner Res 15: Coxon et al 2003, Calcif Tissue Int 72: 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 Geranylgeranylated 0 Rab proteins Ctrl 10µM FTI 5µM10µM FTI FTI 10µM GGTI 5µM 10µM GGTI GGTI

49 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

50 Localisation of BPs and PCs in bone

51 Bisphosphonates have different affinities for hydroxyapatite Nancollas et al 2006, Bone 38: The R 2 side-chain of BPs also contributes to bone affinity HAP Adsorption Affinity Constants at ph KL/10 6 L mol ZL ALN RI 0 CL ETD RI IBN ALN ZL Lawson, Triffitt, Ebetino & Russell ABMR & Davos 2005 & 2006

52 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

53 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 risedronate * * * * etidronate * * * * * * concentration (M) concentration (M) IC 50 values in the nm range! Plotkin, Manolagas, Bellido, Bone 39 (2006)

54 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

55 F-RI localises to osteocyte lacunae in mouse cortical bone osteocyte Vascular channel Green- F-RI Blue-nuclei Red- wheat germ agglutinin

56 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