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WO1993001280A1 - Inhibition de pompe protonique dans des cellules osteoclastiques - Google Patents

Inhibition de pompe protonique dans des cellules osteoclastiques Download PDF

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Publication number
WO1993001280A1
WO1993001280A1 PCT/US1992/005498 US9205498W WO9301280A1 WO 1993001280 A1 WO1993001280 A1 WO 1993001280A1 US 9205498 W US9205498 W US 9205498W WO 9301280 A1 WO9301280 A1 WO 9301280A1
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Prior art keywords
vanadate
proton
pump
sensitivity
atp dependent
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PCT/US1992/005498
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English (en)
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Roland Baron
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Roland Baron
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Publication of WO1993001280A1 publication Critical patent/WO1993001280A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane

Definitions

  • This invention relates to an ATP dependent proton (H + )-pump in osteoclast membranes which is sensitive to both vanadate and NEM, is inhibited by low concentrations of nitrate and contains an immunologically distinct subunit(s) not found in other proton pumps.
  • This unique pump is a potential target for the introduction of various drugs that will inhibit osteoclast bone resorption without affecting functions of other cell types.
  • Osteoclasts and osteoblasts are classes of bone cells responsible for bone resorption and bone formation, respectively.
  • osteoclastic bone resorption is followed faithfully by osteoblastic bone formation at the previous resorption sites.
  • osteoclastic bone resorption must cease.
  • Osteopenia results from an imbalance of the opposing activities of osteoclasts and ostoblasts such that the rate of bone resorption exceeds the rate of accretion.
  • the osteopenia has progressed sufficiently, the bone strength is decreased below the critical fracture point, and a fracture results.
  • the term osteoporosis denotes the occurrence of fractures in osteopenic patients.
  • osteopenia can be treated by manipulating the opposing activities of the osteoclasts and osteoblasts. This reasoning has led to the development of factors that can inhibit the formation and/or activities of osteoclasts and stimulate the formation and/or activities of osteoblasts.
  • Osteoclasts are tightly attached to the bone surfaces that they resorb, and the extracellular compartment between the bone and the ruffled border membrane of the osteoclast is actively acidified by the cell. This
  • ATP-dependent II -pumps play a vital role in acidification, i.e., in the establishment and maintenance of a pH gradient across the membrane.
  • the H+-ATPases of several different organelles have been recently purified and characterized and several of their subunits cloned and sequenced.
  • H-+-ATPase there are three types: 1) The mitochondrial F 0 -F 1 -type ATPase which is composed of several subunits and in inhibited by oligomycin, azide, and efrapeptin; 2) the H+/K+-ATPase, a phosphoenzyme ATPase, composed of two subunits, ⁇ and ⁇ , which is inhibited by vanadate; and 3) vacuolar and secretory granule ATPase, also of a F0-F1 type v/ith multiple subunits, which is inhibited by MEM but not vanadate or mitochondrial ATPase inhibitors.
  • the F0-F1 ATPases are all electrogenic while the
  • H+/K+-ATPase is not.
  • the plasma membrane of yeast and Neurospora crassa contain another type of H+-ATPase, which consists of a single subunit, is
  • the vacuolar-type proton pump is a multisubunit structure formed by the assembly of several different polypeptides, some in one copy and some others in multiple copies. These macro-molecular assemblies are organized in three portions, each with a specific functional role:
  • the catalytic portion of the pump faces the cvtoplasmic side of the plasma membrane and is formed by the assembly of 3 copies of a 70kD subunit (67-72kD), which binds ATP, and 3 copies of a 60kD subunit (57-60kD).
  • the proton transporting portion of the pump is burried in the plasma membrane that is traverses and is composed of several copies of a 16kD, DCCD binding, proteolipid and, probably of one or more copies of a 20kD hydrophobic polypeptide.
  • invention to provide for a uniquely identified ATP dependent proton (H + )-pump in osteoclast membranes.
  • hypercalcemia of malignancy, Paget's disease, arthritis and all other bone and joint diseases which involve excessive bone resorption as a pathogenic mechanism which comprises introducing a suitable therapeutic agent into the unique osteoclast membranes containing the ATP dependent proton (H + )-pump identified herein.
  • the present invention provides for an ATP dependent proton (H + )-pump in osteoclast membranes that is sensitive to vanadate and NEM, is inhibited by low concentrations of nitrate and contains an immunologically distinct subunit(s) not found in other proton pumps.
  • the pump is a potential target for the introduction of various drugs that will inhibit osteoclast bone resorption without affecting functions of other cell types.
  • the identification of a unique subunit(s) that copurifies with the osteoclast vacuolar proton pump presents a potential target for therapeutic intervention in the prevention and treatment of osteopenia and other diseases involving an increased bone resorption.
  • the ATPase is not inhibited by DCCD or nitrate and only slightly inhibited by sulfate.
  • the pump which is the subject of the present invention is only slightly inhibited by sulfate it is very sensitive to DCCD and nitrate.
  • the 115 kD ATPase is not involved in proton uptake into chromaffin granules, whereas in the present case, the vanadate sensitive ATPase is involved in proton transport.
  • osteoclast membrane vesicles Moreover, all groups claim that their osteoclasts membrane preparations are about 70% pure.
  • the inventor in the instant application has
  • osteoclast membrane preparations that have now been isolated can be immunologically distinguished from the preparations used by the other groups working in this field.
  • the other groups observe a protein of 70 kD in their
  • osteoclast membrane preparations that cross reacts with antibodies against a N. crassa 67 kD proton pump subunit.
  • the invention here observes a 70 kD band in his impure fractions that decreases with osteoclast membrane purity.
  • all other groups observe a 60 kD protein in their osteoclast membrane preparations that cross reacts with antibodies raised against an N. crassa 57 kD proton pump subunit. The invention here observed this protein in his impure osteoclast preparations, but the band decreased with purity.
  • the osteoclast proton pump may be a target for therapeutic intervention.
  • the other subunit of the catalytic portion of the osteoclast proton pump 60kD in molecular weight, differs immunologically from that reported by others and is recognized by antibodies to the bovine chromaffin granule but not by antibodies to the N. crassa vacuolar pump, which sees the 60kD subunit in classical vacuolar pumps.
  • Fig. 1.A-1 and 1.A-2 Illustrate highly purified osteoclasts.
  • Fig. 1.B-1 and 1.B-2 Show that the ability of the vesicles to transport protons increases with osteoclast purity.
  • Fig. 1.C Demonstrates the presence of a high density of characteristic F0-F1 ball-and-stalk multisubunit proton pump structures by electron microscopy of negatively stained microsomal vesicles.
  • Fig. 2.A-E Illustrate that the osteoclast proton pump is inhibited by all the classical inhibitors of vacuolar ATPas but also by vanadate, defining it as a new class of proton ATPase, and by low concentrations of nitrate.
  • Fig. 3.A-B Show the immunoblot analysis of the subunit composition of the osteoclast proton pump reveals a new and specific p63 isoform of the vacuolar catalytic 70KD subunit
  • Fig. 4. Shows in Lane A and F: 20 sec phosphorylation in th absence of vanadate; lane B: 20 sec phosphorylation in the presence of 1 mM vanadate; lanes C, D and E: 20 sec
  • Fig. 5 Shows the structure of a classical vacuolar proton pump.
  • Osteoclasts were isolated from medullary bone of calcium deprived laying hens and enriched by unit gravity centrifugation - a procedure routinely used in the laboratory to yield 5-15 ⁇ 10 cells (osteoclasts) per hen of particle purity ranging from 1:4 to 1:7. Though the particle purity of osteoclasts is
  • the purity is 70-90% on the basis of total protein (or membrane) as osteoclasts are ⁇ 30 times larger than the contaminating cells. Furthermore, this level of purity is at least equivalent to that of kidney homogenates previously used by other investigators to purify proton pumps. Other methods of osteoclast isolation, based on percoll or ficoll gradients, have been attempted but they did not significantly improve the purity of the
  • the supernatant from this step was spun at 100,000 ⁇ g for 30 min to get the microsomal
  • This fraction which is highly enriched in the vanadate-sensitive proton transport system, was further fractionated in percoll gradients or by free-flow
  • TEA-sucrose 10 mM triethanol araine, 10mM acetic acid; pH 7.4; 1 mM EDTA and 250 mM sucrose
  • Cells are homogenized with 1-3 passes of a ball bearing homogenizer to obtain > 80% lysis, as monitored by phase-contrast microscopy, leaving nuclei intact and allowing for large sheets of membranes.
  • Microsomes are prepared from a postnuclear supernatant and resuspended to 1 mg protein/ml as previously described. To allow better separation of membranes, microsomes might have to be incubated for 5 min 37°C in 20-25 ⁇ M/ml TPCK-trypsin (Worthington). The digestion is stopped by adding 100 ⁇ M/ml soybean trypsin inhibitor (Sigma) and cooling on ice.
  • the different subcellular fractions are characterized by assaying marker enzymes such as Na + ,
  • ATP-dependent acidification is assayed as previously described and as routinely used in previous work by the applicant, using vesicles isolated from osteoclasts instead of intracellular organelles.
  • the vesicles are diluted in buffer containing
  • Preloading is accomplished by incubating the vesicles in a solution of 0.5 uM valinomycin and 1.5 ⁇ M Acridine
  • osteoclast preparations with vesicles derived from other cell-types or from acidified intracellular orgenelles, the possibility that indeed the pharmacology and structure of the rufged-border osteoclast proton-pump (OC- H+ATPase) could differ from that of other proton pumps but remain undetectable under the experimental conditions used in previous studies, was considered.
  • Osteoclast membranes contain a high number of FO-Fl-like proton pumps
  • microsomal fractions were obtained from osteoclast suspensions at several steps in the purification procedure, ensuring that the pharmacological and structural properties of the proton transport system(s) present in these membranes co-purified with the osteoclasts.
  • osteoclasts were purified 400-fold and plasma membrane enriched another 10-fold after fractionation (Table 1).
  • the ability of the inside-out vesicles present in these membrane fractions to transport protons upon addition of ATP in the acidification assay, expressed as the ⁇ pH/mg protein (Fig. 1B-2) increased proportionally to the purity of the cell preparation, thereby demonstrating that most of the proton transport systems present in the microsomal fraction are derived from osteoclasts.
  • FIG. 1B shows at least 2-fold greater than that of highly purified endosomes (Schmid et al 1989; Fuchs et al 1989).
  • This very efficient H+ transport suggested that a high concentration of pumps was present in these membranes.
  • high magnification electron microscopy on negatively stained osteoclast microsomes was performed (Fig. 1C). 30-40% of the vesicles were found to contain in their limiting membranes high densities of characteristic ball-and-stalk structures, compatible with the presence of high number of copies of F0-F1-ATPases and reminiscent of kidney tubule apical membranes (Brown et al 1987).
  • microsomal preparations v/ere then used to study the properties of proton transport and for
  • the osteoclast membrane F0-F1 proton pump is of a novel type, albeit closely related to the V-ATPases.
  • the main differences found between the OC-ATPase and V-ATPases are 1) Its complete inhibition by both NEM and vanadate; 2) Its complete inhibition by low
  • Fig. 2 Pharmacologically (Fig. 2), all the inhibitors of V-ATPases inhibited 100% of the acidification by osteoclast membrane vesicles.
  • the k1/2 for NEM, DCCD, quercetin and Bafilomycin Al were 0.1 ⁇ M, 35 uM, 3 ⁇ M and 6 nM respectively.
  • ⁇ steoclast-derived microsomal vesicles used in this assay (Fig. 1B), as described on purified kidney membranes (Brown et al 1987).
  • OC-ATPase could also be inhibited by vanadate, which blocked
  • these membranes could contain two type of proton pumps, a V-ATPase conferring the NEM and
  • the PC- H+ ATPase is highly sensitive to nitrate and insensitive to sulfate and acetate
  • the assay allows the identification of the anions whose transport is required, and possible, across the vesicular membranes (here Cl- and Br-, both through chloride conductances).
  • OC-ATPase is vacuolar in nature but has a unique pharmacological and ionic
  • the OC- H+ ATPase differs from other H+ ATPases in its subunit composition
  • Sensitivity to vanadate is a pharmacological property previously found only in P-ATPases (Al-Awqati, 1986). Since antibodies to P-ATPases failed to detect the presence of known P-ATPases in applicant's preparations or in situ., the possibility that the catalytic subunits (67-70 and 57-60kd) of the OC- H+ ATPase, sites where vanadate is known to be competing with phosphate for binding (Arai et al 1987; Macara 1980), could differ from other V-ATPases was tested.
  • OC-ATPase is more closely related to the chromaffin granule 57kd than to the N crassa 60kd subunit, despite their highly conserved sequences (Nelson, and Taiz, 1989).
  • the 70 kd subunit whether detected by antibodies raised against the 70 kd subunits of chromaffin granules (Moriyama, and Nelson, 1988), coated vesicles (Sudhof et al 1989) or N crassa (Bowman et al 1988)
  • the 63kd band was absent from kidney, bone marrow or macrophages (Fig. 3B) but could specifically be induced in bone marrow macrophage cultures in the presence of 1,25-dihydroxyvitamin D3 (data not shown), conditions which induce the expression of several osteoclast gene products (Billecocq et al 1990) .
  • the 70kd, 63kd and 60kd subunits could be
  • osteoclast purification did not induce any decrease in the amount or apparent Mr of the 70kd subunit, nor did it induce the appearance of a 63kd band, and also that the p63 band could be hormonally induced.
  • the OC-H+ATPase therefore, specifically contains a 63kd subunit immunologically related to but distinct from the 70kd subunits of other vacuolar proton pumps. Taken together, these results confirm that nature and specificity of the 63kd as an isoform of the 70kd catalytic subunit of the proton pump which is specifically expressed in the OC-H+ATPase. The fact that the ability of these membranes to transport protons and their sensitivity to vanadate
  • PC-ATPase represents a new class of F0-F1 H+-ATPase, formed by the assembly of several subunits common to other
  • V-ATPases and as least one specific and inducible isoform (p63) of the 70kd catalytic subunit may represent the molecular counterpart for the unique sensitivity of the PC-ATPase to vanadate.
  • the 63kD subunit of the PC-H+ATPase forms a vanadate- sensitive phosphorylated intermediate.
  • microsomal membrane protein was resuspended in 100 ⁇ l of acidification buffer (150 mM KCl, 20 mM HEPES, pH 5.5 with tetramethyl ammonium hydroxide and 5 mM MgSO4) and incubated in the absence (Lanes A and F) or in the presence (Lane C) of 1 mM sodium orthovanadate for 5 min (30).
  • the reaction was started by addition of 200 ⁇ Ci of [ ⁇ 32 P] ATP (50 ul) and stopped after 20 sec by addition of 50 ul of 20% TCA.
  • the reaction was stopped with TCA and the reaction mixture was diluted to 2 ml with 1 mM cold ATP and 1 mM sodium phosphate for 15, 30 or 120 sec.
  • the membranes were pelleted down by centrifugation at 12,000 rpm for 2 min, washed twice and the final pellet was solubilized in 0.5 ml of 10 mM MOPS (ph 7.0), 0.3 M sucrose, 25% glycerol, 2 ug/ml pepstatin A, 5 ug/ml leupeptin and 1% C 12 E 9 .
  • the suspension was vortexed, centrifuged at 12000 rpm for 4 min and the supernatants were incubated 2 hrs at room temperature with the anti N.
  • the novel 63kD subunit also applies to humans.

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Abstract

La présente invention se rapporte à une pompe-(H+) protonique dépendante de l'ATP, identifiée de matière unique dans des membranes d'ostéoclaste. Cette pompe est sensible à la fois au vanadate et au NEM, contient une ou plusieurs sous-unités distinctes d'un point de vue immunologique et que l'on ne trouve pas dans d'autres pompes protoniques, et présente une sensibilité élevée par rapport au nitrate. L'invention se rapporte aussi à un procédé servant à introduire des agents thérapeutiques appropriés dans ladite pompe-(H+) protonique identifiée de manière unique dans des membranes d'ostéoclastes afin de soigner l'ostéopénie et d'autres affections articulaires ou osseuses à activité ostéoclastique accrue (l'arthrose, la maladie de Paget, l'hypercalcémie).
PCT/US1992/005498 1991-07-08 1992-07-07 Inhibition de pompe protonique dans des cellules osteoclastiques WO1993001280A1 (fr)

Applications Claiming Priority (4)

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US72648091A 1991-07-08 1991-07-08
US726,480 1991-07-08
US79609191A 1991-11-20 1991-11-20
US796,091 1991-11-20

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998001443A1 (fr) * 1996-07-09 1998-01-15 Smithkline Beecham S.P.A. Derives d'indole pour le traitement de l'osteoporose

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF BONE AND MINERAL RESEARCH, Volume 5, Number 6, issued 1990, BEKKER et al., "Biochemical Characterization of an Electrogenic Vacuolar Proton Pump in Purified Chicken Osteoclast Plasma Membrane Vesicles", pages 569-579. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 265, Number 13, issued 05 May 1990, SWALLOW et al., "A Vacuolar Type H+-Atpase Regulates Cytoplasmic Ph in Murine Macrophages", pages 7645-7654. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998001443A1 (fr) * 1996-07-09 1998-01-15 Smithkline Beecham S.P.A. Derives d'indole pour le traitement de l'osteoporose
US6903117B2 (en) 1996-07-09 2005-06-07 Nikem Research S.R.L. Indole derivatives for the treatment of osteoporosis

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