EP0968304A1 - Identification d'inhibiteurs de la proteine tyrosine-kinase 2 - Google Patents

Identification d'inhibiteurs de la proteine tyrosine-kinase 2

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Publication number
EP0968304A1
EP0968304A1 EP98906400A EP98906400A EP0968304A1 EP 0968304 A1 EP0968304 A1 EP 0968304A1 EP 98906400 A EP98906400 A EP 98906400A EP 98906400 A EP98906400 A EP 98906400A EP 0968304 A1 EP0968304 A1 EP 0968304A1
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Prior art keywords
pyk2
compound
ability
activity
cells
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German (de)
English (en)
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Le T. Duong
Gideon A. Rodan
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Merck and Co Inc
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Merck and Co Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • This invention is directed to a method of identifying compounds which bind to and/or modulate the activity of the enzyme Protein Tyro sine Kinase (PYK2). Such compounds are useful in the prevention and treatment of osteoporosis and inflammation states.
  • PYK2 Protein Tyro sine Kinase
  • PYK2 Protein Tyrosine Kinase 2
  • CAK ⁇ Cell Adhesion Kinase ⁇
  • RAFTK Adhesion Focal Tyrosine Kinase
  • PYK2 has been cloned from various sources, including mouse, rat and human brain libraries, and the human megakaryocytic CMK cell line.
  • Monocyte-macrophages are migratory phagocytic cells which play an important role in immunity and inflammation, in part due to their capacity to secrete bioactive molecules. Macrophage function is regulated to a large degree by adhesion to surrounding extracellular matrix (ECM) and by responses to specific cytokines.
  • ECM extracellular matrix
  • Monocyte/macrophage adhesion, chemotaxis and phagocytosis are mainly mediated by ⁇ 2 integrins, whose members are classified according to the ⁇ chain as ocL ⁇ 2 (LFA-1; CDlla/CD18), ⁇ M ⁇ 2 (Mac-1; CR3; CDllb/CD18) and ⁇ 2 (gpl50,95; CDllc/CD18).
  • the adhesion of monocytes is also influenced by members of the ⁇ l integrins, particularly 4 ⁇ i (VLA4) and the ⁇ v -associated integrins.
  • podosomes are regions of the cell surface where the plasma membrane is in close contact with the underlying substrate. Podosomes have been detected in many transformed cells but are also extensively present in spreading macrophages and osteoclasts.
  • This invention relates to a method of identifying a compound which binds to and/or modulates the activity of Protein Tyrosine Kinase 2 (PYK2) comprising contacting the compound and PYK2 and determining if binding has occurred. Further, if binding has occurred, the activity of the bound PYK2 may be compared to activity of PYK2 which is not bound to the compound to determine if the compound modulates PYK2 activity.
  • Murine PYK2 cDNA is set forth in Figure 8 (SEQ ID NO:5).
  • a deduced murine PYK2 protein is also set forth in Figure 8 (SEQ ID NO:6).
  • This invention also relates to a method for identifying compounds which inhibit the formation of podosomes in macrophage cells comprising: contacting the compound with protein tyrosine kinase 2 (PYK2) and determining if the compound inhibits PYK2 activity.
  • PYK2 protein tyrosine kinase 2
  • This invention also relates to a method of identifying a compound which prevents monocyte adhesion to a substrate comprising: contacting the compound with protein tyrosine kinase 2 (PYK2) and determining if the compound inhibits PYK2 activity.
  • PYK2 protein tyrosine kinase 2
  • Another aspect of this invention is a method of identifying a compound which inhibits osteoclast mobility comprising: contacting the compound with protein tyrosine kinase 2 (PYK2) and determining if the compound inhibits PYK2 activity.
  • a further aspect of this invention is a method of identifying a compound which inhibits a monocytic cell from degrading an extracellular matrix comprising: contacting the compound with protein tyrosine kinase 2 (PYK2) and determining if the compound inhibits PYK2 activity.
  • the present invention relates to compounds which are identified using the assays of the present invention. The compounds which are identified are useful in the prevention and treatment of osteoporosis, inflammation, and other conditions dependent upon monocyte migration and invasion activities.
  • the present invention also relates to methods of treating and/or preventing a disease state or condition in a mammal which is mediated by PYK2.
  • the present invention also relates to methods of treating and/or preventing osteoporosis and/or inflammation in mammals by administering an effective amount of the compounds which are identified using the assays of the present invention.
  • FIGURE 1 is a Northern Blot analysis of PYK2 expression in mouse tissues. The same RNA blot was hybridized first with a specific probe for mouse PYK2 (upper panel) and then stripped and probed for mouse Focal Adhesion Kinase (FAK) (lower panel) as described. RNA size markers are indicated on the right.
  • FAM Focal Adhesion Kinase
  • FIGURE 2 shows characterization of the polyclonal anti- PYK2 antibodies. Specificity of the anti-PYK2 antibodies was assessed by immunoprecipitation using either anti-PYK2 antiserum (upper panel) or anti-FAK mAb 2A7 (lower panel) from cell extracts either isolated from mock-transfected human embryonic kidney 293 cells (lane 1), or stably transfected with mouse FAK cDNA (lane 2) and mouse PYK2 cDNA (lane 3). The level of expression of each protein was visualized by immunoblot analysis using the respective antibodies.
  • PYK2 and FAK were examined in parental NIH3T3 cells (wt., lane 4), or in Ras-transformed (lane 5) and in Src- transformed (lane 6) NIH3T3 cells. Protein levels of PYK2 (upper panel) and FAK (lower panel) were detected by immunoprecipitation, followed by immunoblot analysis.
  • FIGURE 3 shows expression of PYK2 in murine monocyte-macrophages in primary thioglycolate-induced peritoneal macrophages (lane 1), immortalized peritoneal IC-21 macrophages (lane 2), monocyte-macrophage RAW264.7 (lane 3), WEHI-3 (lane 4) and P388D1 (lane 5) cell lines. Expression of these proteins was also examined in isolated mouse bone marrow cells (lane 6), in bone marrow-derrived M-CSF induced macrophages (lane 7) and bone marrow-derived l,25(OH)2D3 -induced osteoclast-like cells (lane 8). To visualize the doublet on SDS-PAGE that represents the closely separated forms of PYK2, the immunoblot for the levels of PYK2 in these cells was underdeveloped.
  • FIGURE 4 demonstrates that cell adhesion stimulates tyrosine phosphorylation of PYK2 in the IC-21 macrophages in suspension or upon attachment to various ECM.
  • cells in suspension were allowed to attached for 20 min on polystyrene dishes coated with 100 ⁇ g/ml poly-L-lysine (lane 3), 25 ⁇ g/ml fibronectin (FN, lane 4), 10 ⁇ g/ml vitronectin (VN, lane 5), 50 ⁇ g/ml fibrinogen (FB, lane 6), 25 ⁇ g/ml laminin (LN, lane 7), 25 ⁇ g/ml collagen type I (COL I, lane 8) and 25 ⁇ g/ml collagen type IV (COL IV, lane 9).
  • the blot was first incubated with anti-phosphotyrosine mAb 4G10 (upper panel), then stripped and re-blotted with anti-PYK2 antibodies (lower panel).
  • the arrows indicate two forms of PYK2 based on differences in molecular weight.
  • FIGURES 5A, 5B, and 5C demonstrate that adhesion- mediated increase in PYK2 tyrosine phosphorylation is time dependent.
  • IC-21 cells were attached to tissue culture dishes for 4h in the presence of serum (ON DISH, lane 1) or maintained in suspension for lh in serum-free condition (OFF DISH, lane 2). Cells were allowed to attach to fibronectin (FN)-coated dishes in serum-free medium for the indicated times (lanes 3-8).
  • Levels of phosphotyrosine (upper panel) and PYK2 protein (lower panel) were determined by immunoblots using anti-P-Tyr 4G10 mAb and anti- PYK2 Ab, respectively.
  • Figure 5C shows in vitro kinase assays of anti-PYK2 immunocomplexes from IC-21 cells plated on fibronectin (Fn).
  • the bar graph represents the calculated specific activity of total [32p] incorporation into poly(Glu,Tyr) after kinase assays of immunoprecipitated PYK2 complexes, normalized to the protein level as determined by western blots.
  • the activity of PYK2 in ON DISH was arbitrarily set at 1.0, and all numbers represent averages from three experiments.
  • FIGURE 6A demonstrates that adhesion-induced tyrosine phosphorylation of PYK2 is mediated by integrin ⁇ M ⁇ 2 in macrophages. Expression pattern of integrins in IC-21 macrophages was determined by by flow cytometric analysis.
  • mAb M17/4 anti- L
  • mAb Ml/70 anti- ocM
  • niAb HL3 anti- ⁇
  • mAb M18/2 anti- ⁇ 2
  • mAb Rl-2 anti-oc4
  • mAb MFR5 anti- ⁇
  • mAb H9.2B8 anti- ⁇ v
  • mAb 9EG7 anti- ⁇ i
  • Figure 6B shows IC-21 cells which were allowed to attach to fibrinogen in the absence (lane 1) or the presence of blocking antibodies to the following integrin subunits: anti- ⁇ L (lane 2), anti- ⁇ M (lane 3), anti- ⁇ 2 (lane 4), anti- ⁇ 4 (lane 5), anti- ⁇ 5 (lane 6) and anti- ⁇ l (lane 7).
  • Tyrosine phosphorylation of PYK2 was determined by immunoprecipitation and immunoblot analysis.
  • Figure 6C shows that relative PYK2 tyrosine phosphorylation was quantitated in cells adhering to fibrinogen (Fb, upper panel), to fibronectin (Fn, middle panel) and to vitronectin (Vn, lower panel).
  • the specific activity of phosphotyrosine content in PYK2 was determined by normalizing to the protein level.
  • PYK2 tyrosine phosphorylation is expressed relativ ⁇ tb po'ntrtfl (infthe absence of blocking antibodies), which is arbitrarily s ⁇ t at 1.0. Bars represent values from three separate experiments.
  • FIGURE 7 demonstrates that clustering of ⁇ 2-integrin induces tyrosine phosphorylation of ⁇ PYK# i ' macrophages.
  • IC-21 cells were incubated with either anti- ⁇ 2 integrin mAb M18/2 or anti- ⁇ i integin mAb 9EG7. Then, cells were treated with goat F(ab')2 anti- rat IgG (50 ⁇ g/ml) for the indicated times. Cells were lysed and subjected to immunoprecipitation and immunoblot using anti- phosphotyrosine mAb 4G10 and anti-PYK2 antibodies as described.
  • Figure 8 is the cDNA sequence of mouse PYK2 and the deduced protein sequence. Intron sequences are in lower case letters. The exon sequence is capitalized. The boxed sequence of the deduced protein indicates the kinase domain. The circled prolines of the deduced protein indicate the proline rich domain.
  • ECM extracellular matrix
  • COL I collagen type I
  • COL rV is collagen type VI
  • Fb fibrinogen
  • Fn is fibronectin
  • Ln laminin
  • Poly-L-Lys is poly-L-lysine
  • Vn is vitronectin
  • FAK focal adhesion kinase
  • PYK2 is protein tyrosine kinase 2
  • CAK ⁇ is cell adhesion kinase ⁇ ; RAFTK is related adhesion focal tyrosine kinase.
  • PYK2 is the kinase which is primarily responsible for podosome formation in monocyte-macrophages.
  • one aspect of this invention are assays to determine if a candidate molecule can effect PYK2 activity.
  • the assays of this invention which asses a compound's ability to modulate PYK2 activity may be cell-based or may use PYK2 which is no longer in intact cells.
  • PYK2 which is no longer in intact cells.
  • any cell which expresses PYK2 may be used.
  • Such cells and cell lines which naturally express PYK2 are known to those in the art and include: macrophages, osteoclasts, phagocytes, and particularly immortalized mouse peritoneal IC-21 macrophage cell line.
  • any host cell which can be transformed and is capable of transcribing and translating nucleic acids encoding PYK2 may be used.
  • Convenient host cells including mammalian, yeast, and bacterial cells are known to those in the art.
  • the sequence of mouse PYK2 is given in Figure 8 (SEQ ID NO:6).
  • the assays of this invention may be adaptations of any known assay.
  • one embodiment of this invention is a binding assay wherein either the compound to be assayed or PYK2 is labeled. Labels may be chemilumine scent, radioactive, flourescent or any labels routinely used in the art.
  • the compound and the PYK2 are contacted, and incubated for at least a sufficient time for binding to occur. The bound compound-PYK2 can then be separated from unbound compound and unbound PYK2 and the amount of bound entity can be determined by measuring the label.
  • Another assay in accordance with this invention is an i vitro kinase assay.
  • the activity of PYK2 activity is determined by measuring the ability of PYK2 to incorporate a labeled phosphate into a substrate.
  • the phosphate is radiolabeled, and its incorporation into poly-Glutamine or poly-Tyrosine by PYK2 is measured.
  • a potential inhibitor or activating compound is added, and the incorporation rate is compared to the rate of incorporation in the absence of the compound.
  • This assay is exemplified in Example 8.
  • Yet another assay in accordance with this invention measures the ability of PYK2 to phosphorylate itself at tyrosine residue 402. This assay is generally performed using conditions similar to those for the in vitro kinase assay, except that no substrate is required to be present.
  • the incorporation of labeled phosphate into PYK2 is monitored in the presence and absence of the putative inhibitor.
  • the phosphate is radiolabeled and its incorporation into PYK2 is monitored by SDS-PAGE followed by X-ray radiography.
  • the amount of auto-phosphoylation of PYK2 generally reflects the activation state of PYK2.
  • a compound which inhibits autophosphoylation would be a compound which inactivates the kinase.
  • Still another assay in accordance with this invention is an assay which measures the effect (either inhibitory or stimulatory) a candidate compound has an podosome formation in a cell.
  • the cells which may be use in this assay include any cell of interest which is known to form podosomes. If the candidate compound has potential use in osteoporosis, the preferred cell is an osteoclast or osteoclast-like cell.
  • Podosomes are treated by methods known in the art so that they can be visualized, for example, by immunofluorescence. Any inhibitory effect of the candidate compound can then be visually assessed.
  • the above assays which can identify and characterize a compound's ability to inhibit (or activate) PYK2 activity can therefore be used for a variety of endpoints.
  • inhibition of PYK2 can lead to the inhibition of podosome formation, prevention of monocyte adhesion to a substrate, inhibition of osteoclast mobility and inhibition of extracellular matrix degradation, the above assays are useful for identification of compounds with these utilities.
  • IC-21 Adhesion-dependent regulation of PYK2 activity was primarily examined in the immortalized mouse peritoneal IC-21 macrophage cell line.
  • IC-21 shares many characteristics with normal (i.e., non-immortalized macrophages) peritoneal macrophages, including the ability to phagocytize, to secrete lysosomal enzymes, to function as effector cells in antibody-dependent cellular cytoxicity, and to respond to chemoattractants.
  • IC-21 cells possess macrophage-specific antigens, Fc and C3 receptors.
  • Tyrosine phosphorylation of PYK2 is reduced in non- adherent IC-21 macrophages, while cell attachment and spreading on ECM increased PYK2 tyrosine phosphorylation and kinase activity in a time- and substrate-dependent manner.
  • Activation of PYK2 appears to correlate with cell spreading, since macrophages attach to, but spread only slowly on poly-L-lysine, as compared to fibronectin.
  • PYK2 tyrosine phosphorylation proceeded slowly as well.
  • Adhesion-induced PYK2 tyrosine phosphorylation and kinase activation suggests the involvement of integrins in the cell attachment and spreading process. Indeed, clustering of ⁇ 2 integrins, but not ⁇ i integrins, with the respective antibodies induce
  • peripheral blood monocytes migrate to various tissues, where they are known to undergo final differentiation into macrophages.
  • Monocyte migration involves multiple interactions with the endothelial lining, diapedesis between endothelial cells and crossing of the ECM.
  • Podosomes or "rosette" adhesions have been detected in many transformed cells, but they are most abundant in spreading macrophages and osteoclasts.
  • cells which express podosomes have an "invasive" phenotype, they are highly motile and secrete proteases.
  • Podosomes are dynamic structures; they apparently assemble and disassemble within a few minutes. Podosomes have therefore been implicated in the regulation of rapid migration and in local degradation of the ECM. Inhibition of PYK2 activity thus results in reduced motility and decreased matrix degradation in macrophages. Therefore, the podosome-associated PYK2 is a potential crucial intermediate in adhesion-dependent differentiation and activation of macrophages.
  • PYK2 is highly expressed in macrophages and rapidly tyrosine phosphorylated upon cell attachment to specific ECM. This cell adhesion-dependent PYK2 phosphorylation is mediated, in part, by the ligation of integrin ⁇ M ⁇ 2. In addition, PYK2 co-localizes with «M ⁇ 2 to podosomes in macrophages. The localization of PYK2 implicates its function in the formation of podosomes and in the regulation of migration and matrix degradation of monocytic cells. To reach the above conclusions regarding PYK2 function, the following investigations were made.
  • FAK focal adhesion kinase
  • the full length cDNA from a mouse spleen cDNA library was cloned.
  • the deduced amino acid sequence of the full length clone was found to be identical to the recently published amino acid sequence of the mouse RAFTK (Avraham, et al., 1995 supra).
  • full length FAK from a mouse osteoblastic MB1.8 cell line (Wesolowski, et al., 1995 Exp. Cell Res. 219:679-686), and its sequence was the same as that published (Hanks, et al., 1992 Proc. Natl. Acad. Sci. USA 89:8487-8491).
  • PYK2 and FAK cDNAs were subsequently transfected into human embryonic kidney (HEK) 293 cells.
  • HEK human embryonic kidney
  • anti- PYK2 antibodies were developed against the C-terminal domain of mouse PYK2 as described in the Examples, and were affinity purified using the recombinant peptide.
  • the parental HEK 293 cells express endogeneous FAK, but not PYK2 (Fig. 2).
  • Anti-PYK2 antisera recognize a 110 kDa protein in HEK 293 cells transfected with full length mouse PYK2 cDNA (Fig. 2, lane 3), but not the transfected mouse FAK (Fig. 2, lane 2) or the endogenously expressed human FAK (Fig. 2, lane 1-3).
  • PYK2 protein was not detected in NIH-3T3 cells and transformation of this fibroblastic cell line by either v-ras or v-src did not induce PYK2 (Fig. 2, lane 4-6).
  • FAK was not detected in the monocyte-macrophage cell lines, WEHI-3 and P388D1 (Fig. 3, lanes 4 and 5), and in the peritoneal macrophage IC-21 line (Fig. 3, lane 2). FAK was detected in the mouse bone marrow derived macrophage RAW264.7 cell line (Fig. 3, lane 3).
  • PYK2 which is highly expressed in all murine macrophages (Fig. 3, upper panel), appears to present as two forms, differing slightly in molecular weight.
  • the peritoneal macrophage IC21 cells express both forms equally, while WEHI-3 and the bone marrow derived osteoclast-like cells express mainly the higher molecular weight PYK2.
  • RAW264.7, P388D1 the primary peritoneal macrophages and the bone marrow derived macrophages express predominantly the lower molecular weight form of PYK2.
  • PYK2 tyrosine was rapidly dephosphorylated upon trypsinization, but both PYK2 forms are still detected by anti-PYK2 antibodies, as marked by the arrows.
  • Adhesion-dependent PYK2 tyrosine phosphorylation is a rapid response.
  • an increase in PYK2 tyrosine phosphorylation is detected within 1 minute and peaks around 20 minutes after plating (Fig. 5A and 5B).
  • the increase in PYK2 tyrosine phosphorylation upon attachment to fibronectin is associated with a concomittant increase in PYK2 intrinsic kinase activity (Fig. 5C). Attachment to poly-L-lysine caused a much slower increase in PYK2 tyrosine phosphorylation (Fig.
  • integrins ⁇ l and ⁇ 2 integrins are primarily responsible for the adherence of macrophages to other cells and to ECM components such as fibronectin and fibrinogen and ⁇ y integrins mediate binding to vitronectin
  • the surface expression of integrins present in IC-21 macrophages was determined by flow cytometry. As shown in Figure 6A, the predominant integrins are ⁇ 4 ⁇ i and ⁇ M ⁇ 2- Lower levels of integrins ⁇ l and ⁇ L ⁇ 2 were also detected. However, integrins ⁇ 2 and the ay-associated integrins were not detected in this cell line, using mAb HL3 and mAb H9.2B8, respectively.
  • IC-21 macrophages were incubated with blocking antibodies to the ⁇ 2-associated integrin subunits: anti- integrin ⁇ L antibody (M17/4), anti- integrin ⁇ M antibody (Ml/70) or anti- integrin ⁇ 2 antibody (M18/2).
  • blocking antibodies to the integrin subunit ⁇ 4 (Rl-2), or ⁇ 5 (MFR5) and to the integrin subunit ⁇ l (9EG7) were also used.
  • PYK2 tyrosine phosphorylation is specifically inhibited by anti- ⁇ M and anti- ⁇ 2 antibodies when IC-21 cells are seeded on fibrinogen or fibronectin.
  • antibodies to the integrin subunit o4, ⁇ 5 or ⁇ l fail to block the increase in PYK2 tyrosine phosphorylation.
  • antibodies to the ⁇ 2-associated integrins or to the ⁇ i-associated integrins do not prevent the adhesion-mediated PYK2 phosphorylation in IC-21 cells (Fig. 6C).
  • integrin ctM ⁇ 2 is the predominant receptor which mediates IC-21 macrophage attachment to fibrinogen, and regulates cell attachment to fibronectin.
  • IC-21 cells express significant levels of integrin ⁇ 4 ⁇ l and detectable levels of ⁇ L ⁇ 2 > and 05 ⁇ l as shown by flow cytometry (Fig. 6A), however these receptors do not appear to play a significant role in regulating PYK2 phosphorylation during the initial phase within 20 minutes of cell adhesion to fibrinogen and fibronectin.
  • integrin ⁇ M ⁇ 2 The role of integrin ⁇ M ⁇ 2 in mediating PYK2 tyrosine phosphorylation was further supported by integrin ligation.
  • IC-21 macrophages were incubated either with anti-integrin ⁇ 2 antibodies (M18/2) or anti-integrin ⁇ l antibodies (9EG7), and clustering effects were enhanced by incubation with secondary antibodies.
  • PYK2 phosphotyrosine levels were determined after immunoprecipitation by immunoblotting. As shown in Figure 7, ligation of the ⁇ 2 integrin subunit (lane 1-5) increases PYK2 tyrosine phosphorylation, while ligation of the ⁇ l integrin subunit (lane 6) has no effect. Similar to cell attachment, ligation of the ⁇ 2 integrin causes a very rapid (within 5 minutes) increase in PYK2 tyrosine phosphorylation, which remains elevated at least up to 30 minutes at 37°C.
  • the podosome-associated PYK2 was found in the periphery of the ruffled-leading edge of motile cells or organized in extensive arrays, mainly underneath the migrating cell bodies and occasionally under the nucleus. Similar to focal adhesion kinase, podosome associated PYK2 was always found to cluster with proteins highly tyrosine phosphorylated. This is evidence of a role for PYK2 in the assembly and/or disassembly of podosomes in macrophages.
  • PYK2 localizes to podosomes, it may play an important role in anchoring actin filaments.
  • IC-21 macrophages were co-stained with anti-PYK2 antibodies and phalloidin, PYK2 was detected extensively in the perinuclear regions of some cells, where it was never found to associate with F-actin.
  • podosome associated PYK2 (which appeared as dot-like structures) was readily demonstrated to cluster with aggregrates of F-actin.
  • podosome-associated PYK2 was found either at the leading edge or under the lamellipodia and the migrating cell body. Immunostaining of ⁇ -actinin also revealed its co-localization with PYK2 in podosome structure.
  • PYK2 was found to be organized as rings in podosome adhesion contacts in macrophages. This indicates that PYK2 is closely associated with a number of cytoskeletal proteins, including vinculin, talin and paxillin, which were previously identified to form ring-like structures surrounding the actin core in podosomes.
  • IC-21 cells were double stained with anti-PYK2 antibodies and monoclonal antibodies to vinculin, talin or paxillin.
  • PYK2 was again shown to concentrate in podosomes as well as in the perinuclear region. Co-localization of PYK2 with vinculin with talin and with paxillin in the ring-like structure was demonstrated.
  • the ⁇ 2 integrin was previously detected as a diffusion corona of staining around the podosome adhesion contacts in the monocyte-macrophage cell lineage. Since the present functional data suggested the involvement of the integrin ⁇ M ⁇ 2 in the activation of
  • Monoclonal anti-FAK antibody 2A7 was purchased from Upstate Biotech. (UBI, Lake Placid, NY). The following rat anti- mouse ⁇ 2 associated integrins were purified from hybridoma supernatants obtained from the American Type Culture Collection (ATCC, Rockville, MD): mAb M17/4 (anti-aL), mAb Ml/70 (anti- ⁇ M), and mAb M18/2 (anti- ⁇ 2).
  • Rat anti-mouse ⁇ 4 integrin mAb Rl-2 was a gift from Dr. Irving L. Weissman, Stanford University.
  • Monoclonal antibodies to integrin subunits ⁇ 5 (mAb MFR5), ⁇ y (mAb H9.2B8), ⁇ x (mAb HL3) and ⁇ l (mAb 9EG7) were purchased from Pharmingen, San Diego, CA.
  • Antibodies to vincullin (mAb VIN-11-5) and to talin (mAb 8d4) were from Sigma (St. Louis, MO).
  • F(ab)'2 anti-rat IgG, FITC-conjugated goat anti-mouse IgG and TRITC- conjugated donkey anti-rabbit IgG were purchased from Jackson Labs (West Grove, PA).
  • FITC- conjugated goat anti-rat IgG and FITC-conjugated goat anti-hamster IgG were purchased from Boehringer Mannheim Co., (Indianapolis, IN).
  • All horseradish peroxidase (HRP) conjugated secondary antibodies were purchased from Amersham (Arlington Heights, IL), except the direct HRP-conjugated anti-phosphotyrosine mAb 4G10 was from UBI. All secondary antibodies coupled to Sepharose were from Organon Teknika (Durham, NC).
  • IC-21 ATCC, TIB- 186
  • P388D1 ATCC, TIB-63
  • RAW264.7 ATCC, TIB-71
  • WEHI-3 ATCC, TIB-68
  • Murine peritoneal macrophages were prepared as described (Mercurio, et al., 1984, J. Exp. Med. 160:1114- 1125, which is hereby incorporated by reference. Briefly, macrophages were induced by thioglycolate injection into the peritoneal cavities of adult BALB/c mice.
  • Bone marrow derived osteoclast-like cells were prepared as described (Wesolowski, et al., 1995 Exp. Cell Res. 219:679-686, which is hereby incorporated by reference). After the collagenase-dispase treatment, mononucleated tartrate resistant phosphatase positive cells were released from the tissue culture plate using 30 nM echistatin. Freshly isolated osteoclast-like cells were immediately solubilized in immunoprecipitation buffer. EXAMPLE 3
  • mice PYK2 and FAK were initially generated based on the non-homologous region between the proteins, which is adjacent to the C-terminal of the kinase domain.
  • PCR polymerase chain reaction
  • a specific probe for PYK2 (570bp) was generated using the 5'-primer (AGTGA CATTT ATCAG ATGGA G) (SEQ.ID.NO. 1) and the 3'-primer (GAATG GACTG TGCAC CGAGC C) (SEQ.ID.NO.2), with cDNAs of mouse bone marrow derived osteoclast-like cells as template (Wesolowski, et al., 1995, supra).
  • a specific probe for FAK was generated using the following primers: 5'- (CAGCA CACAA TCCTG GAGGA G) (SEQ. ID.NO.3) and 3'- (GCTGA AGCTT GACAC CCTCA T) (SEQ.ID.NO.4) with cDNAs of mouse osteoblastic MB1.8 cells as template (Wesolowski, et al., 1995, supra). These probes were confirmed by sequencing analysis. PYK2 cDNA fragments were cloned from a mouse spleen ZAP II cDNA library (Stratagene, La Jolla, CA) using the specific PYK2 probe.
  • Full length PYK2 cDNA were constructed by ligation of two overlapping clones at the Vspl site.
  • the amino acid sequence of the isolated PYK2 cDNA clone was identical to the previously published mouse RAFTK sequence (Avraham, et al., 1995, J. Biol. Chem. 270: 27742-27751.) .
  • Full length FAK cDNA was generated by PCR according to the published sequence (Hanks, et al., 1992 Proc. Natl. Acad. Sci. USA. 89:8487- 8491, which is hereby incorporated by reference).
  • Both PYK2 and FAK cDNAs were subcloned into pCDNA3 plasmid (InVitrogen, San Diego, CA) and transfected into human embryonic kidney (HEK) 293 cells (ATCC, Rockland, MD) by electroporation at 200V, 960 ⁇ F using a GenePulser (Biorad Labs, Richmond, CA).
  • HEK 293 cells was subsequently subjected to G418 selection (800 ⁇ g/ml, Gibco BRL) and clones were picked after 3 weeks in selection medium.
  • the PYK2 C-terminal domain (from methionine residue 685 to end) was amplified by PCR using the mouse PYK2 as template. Amplified product was cloned into pGEX-4T plasmid (Pharmacia Biotech., Piscataway, NJ) and transformed in E. coli XLl-Blue (Stratagene). Expression of GST-PYK2 C-terminal fragment was induced using 0.5 mM IPTG, purified and cleaved from GST with thrombin, essentially according to the instructions of the manufacturer (Pharmacia).
  • the purified C-terminal fragment of mouse PYK2 was used to immunize two rabbits (Research Genetics, Huntsville, AL) and the titers of both antisera were initially determined by ELISA using the recombinant C-terminal fragment of PYK2. Specificity of the immune sera was subsequently determined by western blot by comparison to the preimmune sera. Polyclonal antibodies were then affinity purified by passing the combined fractions of both antisera through an affinity column, which was constructed using the same purified antigen cross linked to CNBr- activated Sepharose 4B according to the instructions of the manufacturer (Pharmacia).
  • the antibodies were eluted from the column using 0.2 M Glycine, pH 2.5 and ImM EGTA and the eluted fraction was then dialyzed against PBS containning 0.02% azide.
  • Anti-PYK2 antibodies were stored at -70°C at a concentration of 0.5mg/ml.
  • Polystyrene dishes 35 mm, Becton Dickinson, Lincoln Park, NJ were coated overnight at 4°C with either 100 ⁇ g/ml polylysine (Sigma), or 25 ⁇ g/ml human fibronectin (NY Blood Center, New York, NY), or 10 ⁇ g/ml human vitronectin, or 50 ⁇ g/ml human fibrinogen, or 25 ⁇ g/ml mouse laminin (Gibco BRL), or 25 ⁇ g/ml collagen type I or collagen type IV (Collaborative Biomed., Bedford, MA).
  • Antibody-induced clustering in the peritoneal macrophage IC-21 cell line was performed as previously reported (Greenberg, et al., 1994 J. Biol. Chem. 269:3897-3902, which is hereby incorporated by reference). After trypsinization and washing as described above, cell suspensions (1 X 10 ⁇ cells per ml) were incubated with mAb M18/2 or mAb 9EG7 (25 ⁇ g/ml) at 4°C for 30 min.
  • Cells were washed with ice-cold serum free medium (2X) containing 100 ⁇ M sodium vanadate and resuspended in medium containing 50 ⁇ g/ml of goat F(ab)'2 anti-rat IgG and shifted into 37°C incubation for the indicated times. Cells were lyzed in RIPA buffer and subjected to immunoprecipitation and immunoblotting as described below.
  • total cell lysates were prepared by the addition of 1 ml ice cold RIPA buffer containing 1 mM sodium vanadate, 50 mM NaF and a cocktail of protease inhibitors containing 2mM PMSF, 20 ⁇ g/ml aprotinin, 10 ⁇ g/ml leupeptin (Boehringer Mannheim, Indianapolis, IN) and incubated for another 20 min for complete solubilization. After centrifugation, total protein concentration of the clarified lysates was determined.
  • ⁇ g of cell lysates were subjected to immunoprecipitation using either anti-PYK2 antibodies (l ⁇ g) or mAb 2A7 anti-FAK antibody (4 ⁇ g). Immunoprecipitation was carried out for at least 4 hrs at 4°C, followed by addition of anti-rabbit IgG or anti-mouse IgG coupled to Sepharose (Organon Teknika). To study the phosphotyrosine content of PYK2 in IC-21 cells in response to cell adhesion, the attachment assay described above was stopped by addition of an equal volume of 2X ice cold RIPA buffer, and cell lysates were prepared for immunoprecipitation using 2 ⁇ g of anti-PYK2 antibodies.
  • phosphotyrosine was detected by immunoblotting with HRP- conjugated anti-phosphotyrosine mAb 4G10 or with anti-PYK2 polyclonal antibodies, followed by HRP-conjugated anti-rabbit IgG. Blots were developed by enhanced chemiluminescence (ECL, Amersham). ECL signals were determined using an LKB ultroscan XL laser densitometer (LKB, Bromma, Sweden) and the specific activity of tyrosine phosphorylated PYK2 was calculated by comparing the estimated phosphotyrosine contents to protein levels of PYK2. Relative specific activity of phosphorylated PYK2 was normally determined from triplicated experiments.
  • IC-21 cells were solubilized in TNE lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, ImM EDTA, 10% glycerol, 50 mM NaF, 1 mM sodium vanadate and protease inhibitors as described above.
  • PYK2 was immunoprecipitated from the clarified lysates, half of the sample was subjected to immunoblotting with anti-PYK2 antibodies, as described above, and the other half was washed 2 times with the same lysis buffer, and with kinase assay buffer (IX) containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MnCl2 and 1 mM dithiothreitol.
  • IX kinase assay buffer
  • kinase assay buffer containing 5 ⁇ Ci [ ⁇ - 32 P] ATP (3000Ci/mmol, Amersham), 10 ⁇ M ATP, 0.1% BSA and 100 ⁇ g of poly (Glu,Tyr) (molar ratio 4:1; Sigma) was added to the beads and incubated for 10 min at 30°C (Howell and Cooper, 1995 Mol. Cell. Biol. 14:5402-5411).
  • the reaction mixtures (25 ⁇ l) were added to 25 ⁇ l of 30% trichloroacetic acid (TCA) and 0.1 M sodium pyrophosphate, followed by incubation at 4°C for 15 min.
  • TCA trichloroacetic acid
  • the precipitated proteins were transferred to a Multiscreen-FC filter plate (Millipore, Marlborough, MA), washed with ice cold 15% TCA (3X), allowed to dry and incorporation of 32p into the substrate was counted on a Packard top count microplate scintillation counter (Packard, Meriden, CT). Each assay was performed as triplicate. The specific activity was determined by comparing the radioactive counts with immunoblot signals.
  • integrins were analyzed by single- color flow cytometry. After trypsin-EDTA treatment, cells were washed with completed RPMI media containing 10% FBS, twice with Dulbecco's phosphate buffer saline (DPBS) and resuspended in DPBS containing 1% BSA. Cells (2 x 10 ⁇ ) were incubated with the anti- integrins mAbs (2 ⁇ g), as described above, followed by incubation at 4°C for 30 min. The samples were washed once before addition of
  • FITC- labeled goat anti-rat IgG or goat anti-hamster IgG (Boehringer Mannheim). After additional 30 min incubation at 4°C, cells were washed and resuspended in 300 ⁇ l of Flow Cytometric buffer (100 mM Hepes buffer, pH 7.5, 150 mM NaCl, 3 mM KC1 and 1 mM CaCl2) and analyzed by a FACSCalibur (Becton Dickinson, San Jose, CA).
  • Immunofluorescence Microscopy Immunofluorescent labeling of podosomes in IC-21 cells was performed as followed: IC-21 cells were lifted by trypsin-EDTA for 5 min., washed in serum free media (2X), plated on Fn coated glass coverslips and left overnight at 4°C. Cells were washed in PBS (2X) and fixed for 10 min in 4% paraformaldehyde, 2% sucrose in PBS. Cells were then permeabilized in 0.5% Triton, PBS for 5 min, followed by incubation for 1 hr in blocking buffer containing 10% normal goat serum, 1% BSA in PBS. All subsequent incubations with primary and secondary antibodies were performed in the same blocking buffer.
  • PYK2 was visualized using the affinity purified polyclonal anti-mouse PYK2 antibodies, followed by TRITC- donkey anti-rabbit IgG. Actin was stained with 500 mU/ml FITC-phalloidin (Molecular Probes, Inc., Eugence, OR). Phosphotyrosine and paxillin were stained with mouse mAb py20 and mAb 349, respectively. Vinculin and talin were stained using mouse mAb VIN-11-5 and mAb 8d4, respectively. The mouse monoclonal antibodies were visualized using FITC- goat anti-mouse IgG.
  • the integrin subunits L, oc]VL «4 > oc5 > ⁇ l and ⁇ 2 were immunostained using the following rat anti-mouse integrin antibodies: M17/4, Ml/70, Rl-2, MFR5, 9EG7 and M18/2, respectively, followed by FITC- conjugated goat anti-rat IgG. Immunofluorescent labelled cells were photographed through an 100X objective using a Zeiss Axiophot epifluorescence microscope.
  • IC-21 cells were plated on fibronectin-coated glass coverslips in serum-free media. Migrating macrophages with typical fan-like shape were fixed and solubilized. Cells were co- stained for PYK2 using affinity purified anti-PYK2 polyclonal antibodies, followed by TRITC-donkey anti-rabbit IgG, and for phosphotyrosine using mAb py20, followed by FITC-goat anti-mouse IgG. PYK2 appeared as a ring structure in the adhesion contacts, organized in the cell leading edge or in extensive arrays of rosettes under the cell body. The phosphotyrosine appeared as dot-like structures, which predominantly co-localize with PYK2 in macrophages.
  • Podosome-Associated PYK2 co-localized with F-actin in Macrophages IC-21 cells were co-stained with FITC-phalloidin and anti-PYK2 antibodies, followed by TRITC-donkey anti-rabbit IgG.
  • a typical migrating macrophage with a typical fan-like shape or a macrophage with multiple adhesion contacts was chosen.
  • PYK2 localized to perinuclear and dot-like structures at the leading edge or to extensive arrays of podosomes underneath the lamellaepodia.
  • F-actin cores concentrated in podosomes. Co- localization of PYK2 and F-actin was detected in podosomes and tail regions of migrating macrophages while perinuclear PYK2 was not associated with actin filaments.
  • PYK2 Co-localizes with Vinculin, Talin and Paxillin in Podosomes of Macrophages
  • IC-21 cells were co-stained with anti-PYK2 antibodies and with anti -vinculin mAb VIN-11-5, with anti-talin mAb 8d4, and anti-paxillin mAb 349, followed by appropriate conjugated secondary antibodies.
  • PYK2 localized in the perinuclear regions and in podosomes. Only podosome associated PYK2 was co-localized with vinculin, talin and paxillin, which all appear as ring-like structures.
  • IC-21 cells were plated on fibronectin-coated surface and stained with anti-PYK2 antibodies and with rat anti-mouse ⁇ M mAb Ml/70, rat anti-mouse ⁇ 2 mAb M18/2, followed by TRITC-donkey anti- rabbit IgG and FITC-goat anti-rat IgG.
  • MOLECULE TYPE cDNA
  • SEQUENCE DESCRIPTION SEQ ID NO : 5 :
  • AGTGTACCCC TAACGGCCAA GATGGCTTTC TGCATGGACA TTTGAGAGCC AGTATTCCTC 3540
  • CTTTTCTTAC GTCTCCTTTT TCTCCTCCCC
  • CTTTTCACAT CTGCTCCCCT CCTCTCTCAT 3900
  • Val Glu Lys Glu Asp Val Arg lie Leu Lys Val Cys Phe Tyr Ser Asn
  • Trp Met Phe Ala Val Cys Met Trp Glu He Leu Ser Phe Gly Lys Gin 610 615 620

Abstract

La présente invention concerne des dosages de composés qui se lient à la protéine tyrosine-kinase 2 ou qui modulent son activité. Ces ligands sont utilisés pour le traitement de l'ostéoporose et/ou de l'inflammation.
EP98906400A 1997-02-11 1998-02-09 Identification d'inhibiteurs de la proteine tyrosine-kinase 2 Withdrawn EP0968304A1 (fr)

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US3756097P 1997-02-11 1997-02-11
PCT/US1998/002797 WO1998035056A1 (fr) 1997-02-11 1998-02-09 Identification d'inhibiteurs de la proteine tyrosine-kinase 2
US37560 1998-03-09

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EP1009395B1 (fr) * 1997-02-18 2015-04-15 Genentech, Inc. Systeme d'expression de promoteur de bak
AU2209899A (en) * 1998-12-30 2000-07-24 Sugen, Inc. Pyk2 (raftk) and inflammation
EP1630559A3 (fr) * 1998-12-30 2006-06-07 Sugen, Inc. PYK2 (RAFTK) et inflammation
US6861442B1 (en) 1998-12-30 2005-03-01 Sugen, Inc. PYK2 and inflammation
EP1283909A4 (fr) * 2000-05-04 2006-06-07 Univ Yale Reseaux de proteines a haute densite destines au criblage de l'activite de proteines
WO2002038797A2 (fr) * 2000-10-23 2002-05-16 Bristol-Myers Squibb Company Modulateurs de tyrosine kinase de bruton et intermediaires de tyrosine kinase de bruton et leurs procedes d'identification et d'utilisation dans le traitement et la prevention de l'osteoporose et d'etats pathologiques connexes
JP2004530722A (ja) * 2001-06-29 2004-10-07 アブ サイエンス 骨喪失を治療するためのチロシンキナーゼ阻害剤の使用法
CA2452368A1 (fr) 2001-06-29 2003-01-09 Ab Science Nouveaux inhibiteurs selectifs puissants et non toxiques de c-kit
WO2003004007A2 (fr) 2001-06-29 2003-01-16 Ab Science Utilisation d'inhibiteurs de tyrosine kinases pour traiter les maladies intestinales inflammatoires (mii)
ATE330608T1 (de) 2001-06-29 2006-07-15 Ab Science Die verwendung von n-phenyl-2-pyrimidine-amine derivaten zur behandlung von entzündlichen erkrankungen
US7700610B2 (en) 2001-06-29 2010-04-20 Ab Science Use of tyrosine kinase inhibitors for treating allergic diseases
JP2007524374A (ja) * 2003-02-28 2007-08-30 プレキシコン，インコーポレーテッド Ｐｙｋ２結晶構造および使用
JP2008503561A (ja) * 2004-06-21 2008-02-07 ファルマシア・アンド・アップジョン・カンパニー・エルエルシー 骨芽細胞機能を刺激するためのｐｙｋ２阻害薬

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