EP1644031A1 - Inhibitors of the leukocyte prommp-9/beta(2) integrin complex - Google Patents

Inhibitors of the leukocyte prommp-9/beta(2) integrin complex

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Publication number
EP1644031A1
EP1644031A1 EP04742118A EP04742118A EP1644031A1 EP 1644031 A1 EP1644031 A1 EP 1644031A1 EP 04742118 A EP04742118 A EP 04742118A EP 04742118 A EP04742118 A EP 04742118A EP 1644031 A1 EP1644031 A1 EP 1644031A1
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EP
European Patent Office
Prior art keywords
prommp
peptide
binding
mmp
domain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
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EP04742118A
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German (de)
English (en)
French (fr)
Inventor
Michael Stefanidakis
Mikael BJÖRKLUND
Erkki Koivunen
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CTT Cancer Targeting Technologies Oy
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CTT Cancer Targeting Technologies Oy
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Priority claimed from FI20030923A external-priority patent/FI20030923A7/fi
Priority claimed from FI20040616A external-priority patent/FI20040616A0/fi
Application filed by CTT Cancer Targeting Technologies Oy filed Critical CTT Cancer Targeting Technologies Oy
Publication of EP1644031A1 publication Critical patent/EP1644031A1/en
Withdrawn legal-status Critical Current

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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)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6489—Metalloendopeptidases (3.4.24)
    • C12N9/6491—Matrix metalloproteases [MMP's], e.g. interstitial collagenase (3.4.24.7); Stromelysins (3.4.24.17; 3.2.1.22); Matrilysin (3.4.24.23)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/07—Tetrapeptides
    • 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
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70546—Integrin superfamily
    • C07K14/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18

Definitions

  • the present invention concerns peptide compounds, which are inhibitors of integrin- MMP complex.
  • the compounds bind to the CC integrin I domain and inhibit its complex formation with proMMP-9.
  • the compounds thus prevent neutrophil migration, as well as leukocyte migration.
  • the compounds can be used in the treatment of inflammatory conditions, and leukaemia.
  • the leukocyte integrin family consists of four heterodimeric glycoproteins with specific ⁇ -chains (OCL, OC M , oc ⁇ , or OC D ) and a common ⁇ 2 -chain (CD 18). They play an essential role in mediating adhesion of cells in the immune system (1).
  • the major ligand-binding site locates to an 200 amino acid long sequence within the ⁇ -chain called I or inserted domain, which is homologous to the A domains of von Willebrand factor, repeats of cartilage matrix protein and collagen (2).
  • ⁇ 2 integrins M ⁇ 2 s the most promiscuous binder being able to interact with a multitude of unrelated ligands. These include ICAM 1 to 5, complement fragment iC3b, fibrinogen, uPAR, E-selectin and various extracellular matrix proteins (see (3) and references therein).
  • the integrin has also been shown to have a capacity to bind certain enzymes, but whether this is important for leukocyte adhesion or immune reactions is unclear.
  • Such enzymes showing integrin-binding activity are catalase (4), myeloperoxidase (5) and the proteinases elastase (6) and urokinase (7).
  • the peptide inhibitors of the integrin-MMP complex prevent leukaemia cell migration, suggesting a role for the complex in cell motility.
  • the compounds of the invention also attenuated PMN migration in vitro and in vivo, suggesting a role for the MMP-integrin complex in PMN motility.
  • this motif shows a high degree of similarity to the CWDD(G/L)WLC peptide isolated by phage display as an RGD sequence-binding peptide (15).
  • RGD sequence-binding peptide By recognizing the RGD ligand sequence, CWDDGWLC structurally and functionally behaves like a minimal integrin.
  • DDGW peptide in a reverse situation, as a ligand to integrin.
  • the RGD sequence does not compete with the OCM I domain as the GRGDSP peptide at a 1 mM concentration was unable to inhibit proMMP-9 binding to the I domain (unpublished observations of the present inventors).
  • the pepspot analysis indicates that a class of ⁇ 2 integrin ligands contains an active D/E- D/E-G/L-W motif. These include the previously identified ⁇ M ⁇ 2 ligands iC3b, thrombospondin-1, and the enzymes myeloperoxidase and catalase. In our experiments, the peptides derived from several secreted MMPs, but not membrane-bound MT1- MMP, were also active. It is notable that the D/E-D/E-G/L-W motif is relatively conserved in the secreted members of the MMP family.
  • the I domain binding site is located in the vicinity of the zinc-binding catalytic sequence HEFGHALGLDH between the catalytic domain and the fibronectin type II repeats. This location suggests a mechanism for evading proMMP-9 inhibition by tissue inhibitors of MMPs (TIMPs) or ⁇ -macroglobulin. In the absence of inhibitors, the cell surface-localized proMMP-9 would be readily susceptible for activation and substrate hydrolysis, which may also occur in the presence of intact propeptide. On the other hand, because the binding site of the I domain is located in the vicinity of the catalytic groove, it also suggests an explanation for the blocking of MMP-9/ ⁇ 2 integrin interaction by the small molecule MMP inhibitors such as CTT and frihl.
  • the activity of the DDGW peptide in the THP-1 cell migration assay suggests an important function for the integrin-progelatinase complex in leukocyte migration. Obviously, we cannot exclude the possibility that the DDGW peptide blocks binding of other ligands than gelatinases and in this way inhibits the leukocyte migration. However, as the specific gelatinase inhibitor CTT also blocks the THP-1 cell migration, these results strongly suggest that the proMMP-9/ ⁇ 2 integrin complex is the main target for DDGW. Interestingly, the DDGW peptide blocked THP-1 cell migration although it increased the level of proMMP-9 in the medium, which suggests that cell-surface bound rather than total MMP-9 level is a critical factor in cell migration.
  • DDGW and HFDDDE had potent activities in vivo in the mouse peritonitis model, but it is unclear to what extent this was due to inhibition of proMMP-9 as both peptides can potentially inhibit other ⁇ 2 integrin ligands as well.
  • a subset of ⁇ 2 integrin ligands have a DDGW-like sequence and these include, in addition to MMPs, at least complement iC3b and thrombospondin-1.
  • the excellent in vivo activity of DDGW makes it a useful tool to study the components involved in leukocyte migration and the peptide may be considered as a lead to develop anti-inflammatory compounds.
  • Our results suggest that the proMMP-9/oiM ⁇ 2 complex may be part of the neutrophil's machinery for a specific ⁇ 2 integrin-directed movement.
  • the present invention is thus directed to new peptide compounds, in specific to peptide compounds comprising the tetrapeptide motif D/E-D/E-G/L-W.
  • Said compounds can be used as pharmaceuticals, which inhibit leukocyte migration, as well as neutrophil migration.
  • the inhibitory activity was shown both in in vitro and in vivo experiments.
  • the compounds can be used to treat leukaemia and prevent and treat inflammatory conditions.
  • One embodiment of the invention is the use of the compounds of the invention for the manufacture of a pharmaceutical composition for prophylaxis and treatment of conditions dependent on neutrophil migration.
  • Another embodiment of the invention is the use of the compounds of the invention for the manufacture of a pharmaceutical composition for the treatment of conditions dependent on leukocyte migration.
  • a further embodiment of the invention is a pharmaceutical composition
  • a pharmaceutical composition comprising as an active ingredient a compound of the invention, and a pharmaceutically acceptable carrier.
  • a still further embodiment of the invention is a method for therapeutic or prophylactic treatment of conditions dependent on leukocyte or neutrophil migration, comprising administering to a mammal in need of such treatment a leukocyte or neutrophil migration inhibiting compound of the invention in an amount which is effective in inhibiting migration of leukocytes or neutrophils.
  • Specific embodiments of the invention include methods for treatment of leukaemia and inflammatory conditions.
  • APMA aminophenylmercuric acetate
  • ⁇ M ⁇ 2 CD lib/CD 18, Mac-1 integrin
  • CTT CTTHWGFTLC peptide
  • GPA glycolphorin A
  • HMEC human microvascular endothelial cell
  • ICAM intercellular adhesion molecule
  • MMP matrix metalloproteinase
  • NGAL neutrophil gelatinase-associated lipocalin
  • TAT-2 tumor-associated trypsinogen-2; W ⁇ A CTT, CTTHAGFTLC peptide.
  • FIG. 1A to IE Identification of an I domain binding site in progelatinase.
  • FIG. 1A Phage display peptide sequences specifically bound to the ⁇ M I domain. The consensus motif is shown in bold. Peptides with the strongest binding (CILWMDDGW) and the highest similarity (CPEELWWLC) are aligned with human MMPs (accession numbers shown in parenthesis).
  • FIG. IB Phages bearing the CILWMDDGWC peptide or a control peptide were allowed to bind to immobilized CI M I domain-GST fusion protein (20 ng/well) in the absence or presence of 15 ⁇ M DDGW peptide or LLG-C4 peptide. Bound phages were detected using a monoclonal anti-M13 phage antibody. Mean absorbance of triplicate samples ⁇ SD is shown.
  • FIG. IC, ⁇ , M , or ⁇ x I domain-GST fusions were coated on microtiter wells as in B, and the binding of CILWMDDGWC peptide bearing phage or a control phage was measured.
  • FIG. ID Peptides covering the complete sequence of proMMP-9 were synthesized as overlapping peptides on a pepspot membrane.
  • the ⁇ M I domain (0.5 ⁇ g/ml) was allowed to bind to the peptides followed by immunodetection using anti- ⁇ M I domain antibody LM2/1.
  • the ⁇ M I domain-binding peptide 13 (arrow) is shown in boldface and the zinc binding catalytic sequence is underlined.
  • the prodomain (Pro), catalytic domain containing the fibronectin type II repeats (Cat) and hemopexin domain (Pex) are marked to illustrate the domain structure of proMMP-9.
  • FIG. 1 Peptides covering the complete sequence of proMMP-9 were synthesized as overlapping peptides on a pepspot membrane.
  • the ⁇ M I domain (0.5 ⁇ g/ml) was allowed to bind to the peptides followed by immunodetection using anti- ⁇ M I domain antibody LM2/1.
  • FIG. 2A to 2C Binding of progelatinases to purified integrins and I domains.
  • FIG. 2B binding of proMMP-9 (80 ng/well) was examined on microtiter wells coated with an integrin ( ⁇ 2, 0 M ⁇ 2 , ⁇ i ⁇ i, ⁇ 3 ⁇ or an I domain ( ⁇ L , ⁇ M, ⁇ x). The binding was determined using anti-MMP-9 antibody.
  • FIG. 2C proMMP-2 (80 ng/well) was allowed to bind to ⁇ L ⁇ 2, 0CM ⁇ 2, ⁇ i ⁇ i or the I domains ⁇ L , ⁇ M or ⁇ x. The binding was determined using an anti-MMP-2 antibody.
  • FIG. 3A to 3D Inhibitors of the proMMP-9 / ⁇ 2 integrin complex.
  • FIG. 3A ⁇ M and ⁇ I domain-GST fusions were immobilized on microtiter wells.
  • ProMMP-9 100 ng/well was added in the presence or absence of various peptides (200 ⁇ M) or lovastatin (100 ⁇ M) in a buffer containing 0.5% BSA.
  • ProMMP-9 binding was detected using a monoclonal antibody against MMP-9. The results are shown as percent binding c ompared t o b inding i n t he absence o f i nhibitors ( 100%) a nd no p roMMP-9 added (0%).
  • FIG. 3B DDGW peptide blocks proMMP-9 binding to ⁇ M in a dose dependent manner.
  • the assay was done similarly as in (A), except various concentrations of peptides were added to compete for binding. All samples were assayed as triplicates and results shown are means ⁇ SD from a representative experiment.
  • FIG. 3C proMMP-9 binding to ⁇ M ⁇ 2 and ⁇ L ⁇ 2 was examined in the absence and presence of EDTA (5 mM), MMP inhibitor-1 (100 ⁇ M), CTT (200 ⁇ M), STT (200 ⁇ M), MEM170 (40 ⁇ g/ml), or control TL3 antibody (40 ⁇ g/ml).
  • the background of primary and secondary antibodies was measured by omitting proMMP-9 from the wells or by coating with ICAM-1.
  • FIG. 3D proMMP-9 binding to purified ⁇ M and ⁇ L I domain GST fusion proteins or wild type GST was studied in the absence or presence of competitors as indicated.
  • Control shows the background when proMMP-9 was omitted.
  • FIG. 4A and 4B Coprecipitation of progelatinase with ⁇ 2 integrin.
  • FIG. 4A ⁇ M ⁇ 2 integrin (3 ⁇ g) was incubated with a 500 ⁇ l sample of HT1080 medium containing proMMP-9 and proMMP-2 in the absence or presence of CTT or STT (200 ⁇ M) for 2 h.
  • the integrin was immunoprecipitated with the OKM10 antibody, and the immunoprecipitates were analyzed by gelatin zymography. In control experiments, integrin was omitted from the medium and ICAM-1 was added instead.
  • FIG. 4B a 500 ⁇ l sample of HT1080 medium containing proMMP-9 and proMMP-2 was incubated with the ⁇ M I domain GST (3 ⁇ g) or LLG-C4-GST control.
  • ICAM-1 ICAM-1
  • LM2/1, CTT, STT, or LLG-C4 were used as competitors.
  • GST was pulled down with glutathione beads, and bound proteins were analyzed by zymography.
  • the lane 1 in the figure insert the proMMP-2 and proMMP-9 zymogens present in non-treated HT1080 medium
  • lane 2 lack of gelatinases pulled down with control LLG-C4-GST
  • lane 3 proMMP-9 and proMMP-2 coprecipitated by ⁇ M I domain GST fusion protein.
  • FIG. 5A and 5B CTT peptide binds to both latent and active MMP-9.
  • FIG. 5A Binding of proMMP-9 or APMA-activated MMP-9 to CTT-GST was examined in the absence or presence of competitors CTT (100 ⁇ M), W ⁇ A mutant CTT (100 ⁇ M), and Inhl (100 ⁇ M). GST control was LLG-C4-GST. Binding was determined as in Figs. 2 and 3. The background in the absence of proMMP-9 is shown.
  • FIG. 5B THP-1 cells were incubated in serum-free medium containing CTT, Inhl or W ⁇ A CTT at 200 ⁇ M concentration. Samples from the media were collected at the time points indicated and analyzed by zymography (panels 1, 3, and 4) or Western blotting (panel 2).
  • FIG. 6 A to 6 C Progelatinases o ccur i n c omplex w ith ⁇ M ⁇ 2 a nd ⁇ L ⁇ 2 i n P DBu- activated THP-1 and Jurkat cells.
  • FIG. 6A THP-1 cell surface proteins were [ 3 H]-labelled using periodate-tritiated borohydride and analyzed by immunoprecipitation. CTT was used as a competitor (200 ⁇ M). The immunoprecipitated samples were resolved on a 8-16% polyacrylamide gel, and the film was exposed for 3 days. Lanes 1-4 are from non-activated cells and lanes 6- 10 from PDBu-activated cells. Lane 5 shows molecular weight markers.
  • FIG. 6A THP-1 cell surface proteins were [ 3 H]-labelled using periodate-tritiated borohydride and analyzed by immunoprecipitation. CTT was used as a competitor (200 ⁇ M). The immunoprecipitated samples were resolved on a 8-16% polyacrylamide
  • lysates from PDBu-activated THP-1 cells were immunoprecipitated with integrin or MMP antibodies followed by Western blotting with ⁇ M (OKM10), ⁇ L (TS2/4) or MMP-9 antibodies. Preclearings of the cell lysates were done using ⁇ M (lane 6) and ⁇ L (lane 7) antibodies.
  • FIG. 6C lysates from PDBu-activated Jurkat cells were subjected to immuno- precipitation followed by blotting with the ⁇ L (MEM83) and MMP-9 antibodies.
  • FIG. 7A and 7B PDBu-induced colocalization of ⁇ M ⁇ 2 and proMMP-9 in THP-1 cells. Cells were preincubated for 30 min at +37°C with 50 nM PDBu.
  • FIG. 7A cells were treated with anti- ⁇ M OKM10 and anti-MMP-9 antibodies followed by FITC-labeled (green fluorescence) and TRITC-labeled (red fluorescence) secondary antibodies. Yellow color indicates colocalization of ⁇ M ⁇ 2 and proMMP-9. Bars, 8.5 ⁇ m.
  • FIG. 7B immunofluorescence staining shows intense colocalization of MMP-9 (polyclonal antibody) and ⁇ M ⁇ 2 integrin (OKM-10) on the surface of PDBu-activated THP-1 c ells at higher magnification as visualized by e onfocal microscopy (Bars, 2.5 ⁇ m).
  • the D DGW p eptide s upports T HP-1 c ell a dhesion a nd i nduces proMMP-9 release, but does not block adhesion to the major ⁇ 2 integrin ligands fibrinogen and ICAM-1.
  • FIG. 8 A THP-1 cells were allowed to bind to immobilized, glutaraldehyde polymerized peptides with or without phorbol ester activation (50 nM) and the adherent cells were quantitated by phosphatase assay. THP-1 cells were allowed to bind to immobilized fibrinogen (in FIG. 8B), or recombinant ICAM-1 -Fc (in FIG. 8C), in the presence or absence of 200 ⁇ M soluble peptides. All samples were assayed as triplicates and results show means ⁇ SD. Identical results were obtained in two other independent experiments.
  • FIG. 8D THP-1 cells were incubated in the presence or absence of peptides at 200 ⁇ M concentration for 48 hours. Aliquots of conditioned medium were analyzed by gelatin zymography. Arrows show the 92 kDa proMMP-9 and 220 kDa proMMP-9 dimer.
  • FIG. 9A to 9C Peptide inhibition of THP-1 cell migration.
  • THP-1 cells were preincubated with the peptide as indicated at a 200 ⁇ M concentration for 1 h at room temperature and applied to transwells in the absence (FIG. 9A), or presence (FIG. 9B), of LLG-C4-GST coating. Cells were allowed to migrate for 16 hours at +37°C. Cells migrated to the lower surface of the filter were stained and counted microscopically.
  • FIG. 9C HT1080 fibrosarcoma cell migration was similarly assayed in the absence of LLG-C4-coating. The bars show means ⁇ SD from triplicate wells.
  • FIG. 10A to 10D ⁇ M -I domain binding to recombinant MMP-9 domains.
  • FIG. 10A Schematic representation of MMP-9 and its recombinant forms produced in E. coli.
  • FIG. 10B ProMMP-9, its recombinant forms or BSA were coated on microtiter wells
  • FIG. 10C Binding of proMMP-9 to the immobilized GST- ⁇ M I domain was studied in the presence of each peptide at the concentrations indicated. The binding was determined with the anti-MMP-9 antibody G ⁇ -213.
  • FIG. 10D Binding of GST- ⁇ M I domain to the immobilised proMMP-8, proMMP-9,
  • ICAM-1, and fibrinogen was studied with ICAM-1, DDGW or KKGW (50 ⁇ M) as competitors.
  • GST was added instead of GST- ⁇ M I domain. The experiment was repeated three times with similar results.
  • FIG. 11A to 11D Recognition of recombinant MMP-9 domains by a M p 2 integrin- expressing cells.
  • the studied cells were PMNs (11A, 11B, 11C), a M ⁇ 2 L-cell transfectants (11D), non-transfectants (11D), and LAD-1 cells (11D).
  • PMNs were in resting state or stimulated with PMA (11A, 11C) or C5a or TNF ⁇ (11B) before the binding experiment to proMMP-9 or its domains.
  • Cells were also pretreated with each peptide (50 ⁇ M), antibody (20 ⁇ g/ml) or the ⁇ M I domain as indicated. Unbound cells were removed by washing and the number of adherent cells was quantitated by a phosphatase assay. The experiment was repeated three times with similar results.
  • FIG. 12A to 12 D Blockage of PMN and THP-1 cell migration in vitro by gelatin- ase and ⁇ 2 integrin inhibitors.
  • PMNs (lxlO 5 in 100 ⁇ l) were applied on the LLG-C4- GST or GST coated surface (12A) or HMEC monolayer (12B) in the absence or presence of peptides (200 ⁇ M) or antibodies (20 ⁇ g/ml) as indicated.
  • PMNs were stimulated with 20 nM PMA (12A), HMECs with 50 ⁇ M C5a or lOng/ml TNF ⁇ or left untreated (12B).
  • THP-1 cells (5xl0 4 in lOO ⁇ l) were stimulated with 50 nM PMA and applied on the coated surfaces together with each peptide (200 ⁇ M) (12C). The cells migrated through transwell filters were stained and counted microscopically. All experiments were repeated at least twice.
  • FIG. 13A to 13D Inhibition of neutrophil migration to an inflammatory tissue.
  • FIG. 13A Mice were injected with thioglycolate or PBS intraperitoneally. The peptides were applied intravenously at the amounts indicated. After 3 h, the intraperitoneal leukocytes were harvested and counted. The results show means ⁇ SD of 2-4 mice in a group. (*) indicates statistical significant difference (pO.OOl). The experiment was repeated at least 3 times. The infiltrated neutrophils of mice treated with thioglycolate (13B) or PBS (13C) were stained with anti-MMP-9 and anti- ⁇ M by incubating the cells with the antibodies for 3 h.
  • FIG. 13D Gelatinolytic activity of the supernatants from the peritoneal cavities of mice collected as in (13A).
  • Lanes 1-4 samples are from thioglycolate-treated mice; lane 5: a sample from PBS-treated mouse.
  • DDGW, HFDDDE, and DFEDHD were injected intravenously at doses of 0.1, 0.2 and 0.2 mg per mouse.
  • the arrows show proMMP-9 dimer, proMMP-9 and proMMP-2. The experiment was repeated three times with similar results.
  • the antibodies MEM170 and LM2/1 were against the ⁇ M and the MEM-83 and TS2/4 antibodies against the ⁇ L integrin subunit (19, 20).
  • the monoclonal antibody 7E4 (21) reacted with the common ⁇ 2 -chain of the leukocyte integrins.
  • the ⁇ M antibody OKM10 was obtained from the American Type Culture Collection, ATCC, Rockville, MD (22).
  • a monoclonal antibody against ICAM-5 (TL3) (23) was used as an antibody control.
  • the monoclonal anti-MMP-9 antibody (GE-213) and anti-MMP-2 antibody (Ab-3) were obtained from Lab Vision Corporation (Fremont CA) and from OncogeneTM research products, respectively.
  • Affinity purified rabbit anti-MMP-9 polyclonal antibodies were from the Borregaard laboratory (24). As monoclonal antibody controls, we used a mouse I gG (Silenius, Hawthorn, Australia) and anti-glycophorin A (GPA) (ATCC). Anti-trypsinogen-2 (TAT-2) antibody was a rabbit polyclonal antibody control (32). The peroxidase-conjugated anti-GST mAb was from Santa Cruz Biotechnology. The rabbit anti-mouse horseradish peroxidase-conjugated secondary antibody was from Dakopatts a/s (Copenhagen, Denmark).
  • Inhl (2R-2-(4- biphenylylsulfonyl)amino-N-hydroxy-propionamide) was purchased from Calbiochem, La Jo 11a, CA.
  • Human recombinant ICAM-1 was obtained commercially by R&D systems (Minneapolis, MN).
  • ICAM-1 -Fc fusion protein was expressed in Chinese hamster ovary cells and purified as described (14).
  • the synthetic peptides CTT, STT, LLG-C4 and RGD-4C were obtained as previously described (14, 25). W ⁇ A CTT was ordered from Neosystem, France.
  • ProMMP-2 and proMMP-9 were obtained commercially (Roche).
  • the commercial proMMP-9 showed the 92kDa monomer, 200 kDa homodimer, and 120 kDa NGAL complex bands.
  • the integrins ⁇ 1 ⁇ 1 and ⁇ 3 ⁇ i were purchased from Chemicon International (Temecula, CA). Human plasma fibrinogen and lovastatin were from Calbiochem.
  • Phage display selections were made using a pool of random peptides CX 7-10 C and X 9-1 o, where C is a cysteine and X is any amino acid (14, 25). Briefly, ⁇ M I domain-GST or GST fusion protein was immobilized on microtiter wells at 20 ⁇ g/ml concentrations and the wells were blocked with BSA. The phage library pool was first subtracted on wells coated with GST and then unbound phage was transferred to ⁇ M I domain-GST-coated wells in 50 mM Hepes/5 mM CaCl 2 /l ⁇ M ZnCl 2 /150 mM NaCl/2% BSA (pH 7.5). After three rounds of subtraction and selection, individual phage clones were tested for binding specificity and the sequences of the phage that specifically bound to the I domain were determined (14).
  • the phage peptides were initially prepared biosynthetically as intein fusions.
  • the DNA sequences encoding the peptides were PCR cloned from 1 ⁇ l aliquots of the phage- containing bacterial colonies that were stored at -20 C.
  • the forward primer was 5'-
  • ACTTTCAACCTGCAGTTACCCAGCGGCCCC-3' The PCR conditions included initial denaturation at 94°C for 2 min followed by 30 cycles of 94°C 30 sec, 55°C 30 sec, and 72°C 30 sec.
  • the PCR products were purified using QIAGEN Nucleotide removal kit. They were then digested with Sapl and Pstl restriction enzymes and ligated to a similarly digested and phosphatase treated pTWTN vector (New England Biolabs).
  • Intein fusion proteins were produced in E. coli strain ER2566 and affinity purified on a chitin column essentially as described (26). The peptide was cleaved on the column, eluted and finally purified by
  • Phage 10 8 infective p articles/well) in 50 mM Hepes/5 mM C aCl 2 /l ⁇ M ZnCl 2 /0.5% BSA (pH 7.5) were added to microtiter wells coated with I domain-GST fusion or GST (20 ng/well). The phages were allowed to bind in the absence or presence of a competitor peptide (15 ⁇ M) for one hour followed by washings with PBS containing 0.05% Tween 20.
  • the bound phage was detected using 1 :3000 dilution of a peroxidase- labelled monoclonal anti-phage antibody (Amersham Biosciences) and o- phenylenediamine dihydrochloride as a substrate. The reactions were stopped by addition of 10% H 2 SO 4 and the absorbance was read at 492 nm using a microplate reader.
  • the peptides were synthesized on cellulose membranes as described (27). The membrane was blocked with 3% BSA in TBS containing 0.05% Tween 20, and incubated with 0.5-5 ⁇ g/ml ⁇ M I domain for 2 h at room temperature. The DDGW peptide was used as a competitor at a 50 ⁇ M concentration. Bound ⁇ M I domain was detected using the monoclonal antibody LM2/1 (1 ⁇ g/ml) or MEM-170 (5 ⁇ g/ml) and peroxidase-conjugated rabbit anti-mouse antibody (1:5000 dilution) followed by chemiluminescence detection.
  • the human HT1080 fibrosarcoma and THP-1 and Jurkat leukemic lines were obtained from ATCC and maintained as described previously (20, 25, 28).
  • OCI/AML-3 derived from the primary blasts of an AML patient (29) was maintained in 10% FBS/RPMI supplemented with L-glutamine, penicillin and streptomycin.
  • Cell viability was assessed with a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay according to the instructions of the manufacturer (Roche).
  • integrins ⁇ L ⁇ 2 (CDlla/CD18, LFA-1), ⁇ M ⁇ 2 (CDllb/CD18, Mac-1) and ⁇ x ⁇ 2 (CDllc/CD18) integrins were purified from human blood buffy coat cell lysates by adsorption to the anti-CDl la (TS 2/4), anti-CDllb (MEM170), or anti-CDllc (3.9) antibodies linked to protein A-Sepharose CL 4B.
  • the integrins were eluted at pH 11.5 in the presence of 2 mM MgCl 2 , and 1% n-octyl glucoside as described previously (28). Expression and purification of GST fusion proteins
  • the ⁇ L , ⁇ M , and ⁇ I domains were produced as GST fusion proteins in E. coli strains BL 21 or JM109 and purified by affinity chromatography on glutathione-coupled beads (30, 31).
  • GST containing CTT in the C-terminus was constructed using the protocols described for LLG-C4-GST (14) and glutathione-coupled beads were employed for purification.
  • the purity of the GST-fusion proteins was confirmed by SDS-PAGE with Coomassie Blue staining and Western blot analysis. For pepspot analysis, GST was cleaved from the ⁇ I domain with thrombin.
  • the purified I domains (GST- ⁇ M, GST- ⁇ L, GST- ⁇ ), or integrins ( ⁇ M ⁇ 2, ⁇ L ⁇ 2 , oc ⁇ 2 , ⁇ i ⁇ (1 ⁇ g/well) were immobilized in 20 mM Tris, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , and 1 mM MnCl 2 , pH 7.4.
  • the wells were washed with PBST (10 mM phosphate, 140 mM NaCl, pH 7.4, containing 0.05% Tween20) and blocked with 3% BSA in PBST.
  • ProMMP-2, proMMP-9, or the p-aminophenyl mercuric acetate (APMA) or trypsin-activated forms (32) were incubated for 2 h at room temperature.
  • CTT and Inhl were first preincubated with the proMMPs for 30 minutes at room temperature.
  • the wells were washed three times and incubated with anti-MMP-9 (GE-213) or anti-MMP-2 (Ab-3) antibody at a 2 ⁇ g/ml concentration in PBST for 1 h.
  • Bound antibodies were detected using peroxidase-conjugated rabbit anti- mouse IgG (DAKO, Glostrup, Denmark) and o-phenylenediamine dihydrochloride as a substrate.
  • Serum-free conditioned m edium containing proMMP-2 and proMMP-9 was collected from human HT-1080 fibrosarcoma cells grown in the presence of 100 nM phorbol ester 4 ⁇ -Phorbol 12,13-dibutyrate (PDBu) (Sigma-Aldrich, St. Louis, MO) overnight at +37°C. A 500 ⁇ l volume of the supernatant was incubated with 1 00 ng of GST- ⁇ M , GST- ⁇ L , or GST- ⁇ x I domain or ⁇ M ⁇ 2 integrin for 3 h at 25 °C. GST and GST-LLG-C4 were used to determine non-specific binding.
  • PDBu phorbol ester 4 ⁇ -Phorbol 12,13-dibutyrate
  • CTT, STT, LLG-C4, and ICAM-1 were used as competitors at a 200 ⁇ g/ml concentration, and the antibodies LM2/1 and TL3 at 40 ⁇ g/ml.
  • complexes of I domain and gelatinases were pelleted with Glutathione Sepharose. Integrin complexes were captured by incubating first with the OKM10 antibody for 3 h at +4°C and then with protein G Sepharose for 1 h. After centrifugation and washing, samples were analyzed by gelatin zymography on 8% SDS-polyacrylamide gels containing 0.2 % gelatin (32).
  • THP-1 cells (40 000/100 ⁇ l) were incubated in serum-free RPMI medium for 48 h in the absence or presence of 200 ⁇ M p eptide as described in the text. Aliquots o f the conditioned media were analyzed by gelatin zymography.
  • CTT-GST and GST control (5 ⁇ g/well) were coated overnight on 96-well microtiter plates in 50 ⁇ l TBS followed by blocking of the wells by BSA.
  • proMMP-9 or APMA- activated form (80 ng/well) was incubated in the absence or presence of competitors for 2 h in 50 ⁇ M Hepes buffer containing 1 % BSA, 5 mM CaCl 2 , and 1 ⁇ M ZnCl 2 (pH 7.5). After washing, bound MMP-9 was determined with anti-MMP-9 and HRP- conjugated anti-mouse IgG as described above.
  • THP-1 cells were activated with PDBu for 30 min and then incubated with CTT, W ⁇ A CTT, or Inhl (each 200 ⁇ M) at +37°C in serum-free medium. Samples were taken from the media at 0, 1, 2, 3, 4, and 5 h time points and analyzed by zymography and Western blotting with polyclonal anti-MMP-9 antibodies. Experiments with HT-1080 cells were performed similarly except that the medium samples were collected after 6 h.
  • Non-activated or PDBu-activated THP-1 cells (1 x 10 7 ) were subjected to surface labelling using periodate tritiated sodium borohydride (33).
  • the [ 3 H]-labelled cells were lysed w ith 1 % ( v/v) Triton X-100 in PBS, c larified b y centrifugation a nd p recleared with protein G-Sepharose.
  • the lysate was immunoprecipitated with polyclonal anti- MMP-9, ⁇ M (OKM-10) or ⁇ 2 (7E4) antibodies.
  • Immunodetection was performed with ⁇ M (MEM 170) antibody (10 ⁇ g/ml) followed b y p eroxidase-conjugated anti-mouse IgG and chemiluminescence d etection (Amersham Biosciences). The membranes were stripped of bound antibodies and reprobed with monoclonal ⁇ L chain (TS2/4) or polyclonal anti-MMP-9 antibodies.
  • Immunofluorescence was performed on resting cells or the cells activated with PDBu for 30 min. A portion of the cells was treated with ICAM-1 or CTT to block ⁇ 2 integrins or gelatinases, respectively. Cells were bound to poly-L-Lysine coated cover slips, fixed with methanol for 10 min at -20°C or with 4% paraformaldehyde for 15 min at +4°C, and permeabilized with 0.1% Triton X-100 in PBS at room temperature for 10 min followed by several washings. The cover slips were incubated with rabbit anti-MMP-9 polyclonal and mouse anti- ⁇ M (OKM-10) antibodies diluted 1:500.
  • the secondary antibodies rhodamine (TRITC)-conjugated p orcine anti-rabbit or FITC-conjugated goat anti-mouse (Fab') 2 (Dakopatts a/s, Copenhagen, Denmark) were incubated at a 1:1000 dilution for 30 min at room temperature.
  • the samples were mounted with moviol, incubated in the dark for 2 days, and examined by a confocal microscope (Leica multi band confocal imagine spectrophotometer) at a 400x magnification or a fluorescence microscope (Olympus Provis 70) at a 60x magnification.
  • PMNs were isolated from peripheral blood anticoagulated in acid-citrate dextrose. Erythrocytes were sedimented by centrifugation on 2% Dextran T-500, and the leukocyte-rich supernatant was pelleted, resuspended in saline and centrifuged on a Lymphoprep (Nyegaard, Oslo, Norway) at 400g for 30 minutes to separate polymorphonuclear c ells from p latelets a nd m ononuclear c ells ( 16). P MN p urity was >95% with typically ⁇ 2% eosinophils. Cell viability was measured using an MTT (3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium) bromide assay as instructed by the manufacturer (Roche).
  • MTT 3- [4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium
  • HMEC-1 Human microvascular endothelial cells (17), kindly provided by S. Mustjoki (Haartman Institute, University of Helsinki), were grown in RPMI 1640 in the presence of 10% FBS containing 2 mM glutamine, 100 IU/ml penicillin and 100 ⁇ g/ml streptomycin. Human m onocytic T HP- 1 c ells w ere m aintained a s d escribed ( 14, 25).
  • LAD-1 Leukocyte adhesion deficiency type-1
  • LAD-1 cells, wild type and ⁇ M ⁇ 2 -transfected L929 mouse fibroblastic cells were generous gifts from Dr. Jean-Pierre Cartron (INSERM, Paris, France). These cells were maintained as described previously (18) and the ⁇ M ⁇ 2 expression was examined by fluorescence-activated cell sorting (FACS, Becton Dickinson, San Jose, CA).
  • Fibrinogen and ICAM-1 -Fc were coated at 40 ⁇ g/ml in TBS at +4°C.
  • Peptides (2 ⁇ g/well) were coated in TBS containing 0.25% glutaraldehyde at +37°C.
  • the wells were blocked with 1% BSA in PBS.
  • THP-1 cells (50 000/well) with or without PDBu activation were added in 0.1% BSA-RPMI medium in the presence or absence of 200 ⁇ M peptides or monoclonal antibodies at 50 ⁇ g/ml. After 30-35 minutes the wells were washed with PBS to remove non-adherent cells and the adhesive cells were quantitated by a phosphatase assay.
  • the cell migration assay was conducted using transwell migration chambers (8 ⁇ m pore size, Costar) in serum-containing medium as described (14). Briefly, the membranes were coated on the upper and lower surface with 40 ⁇ g/ml GST, LLG-C4-GST, or left uncoated. The wells were blocked with 10% serum- containing medium for 2 h. THP-1 cells (50 000/100 ⁇ l) or HT1080 (20 000/100 ⁇ l) were preincubated with the peptides for 1 h before transfer to the upper chamber. The lower chamber contained 500 ⁇ l of the medium without the peptides. The cells were allowed to migrate to the lower surface of the membrane for 16 h and then stained with crystal violet and counted.
  • MMP-9 proteins (200 nM in PBS) were coated at +4°C for 16 h and the microtiter wells were blocked with 3 % BSA in PBS for 1 h at room temperature.
  • the ⁇ M ⁇ 2 -integrin L- cell transfectants and PMNs (lxl 0 5 cells/well) were suspended in RPMI medium supplemented with 2mM MgCl 2 and 0.1% BSA and activated with PMA (20nM) for 20 min, or with C5a (50nM) or TNF- ⁇ (lOnM) for 4h at +37°C.
  • the L926 wild type and LAD-1 cells were used as controls.
  • the cells were treated with the indicated antibody (20 ⁇ g/ml) or peptide (50 ⁇ M) at +37°C for 30 min, washed twice with serum-free medium and incubated in the microtiter wells at +37°C for 30 min. The wells were washed with PBS, and the number of adherent cells was quantitated by a phosphatase assay (14). Cell migration was conducted using Costar 24-transwell migration chambers with a 3 ⁇ m pore size for PMNs and 8 ⁇ m for THP-1 cells.
  • HMECs 4xl0 5 cells/well
  • culture medium was changed after 3 days.
  • chemotactic activation was carried out by adding C5a (50nM), TNF- ⁇ (lOng/ml), or medium alone to the lower compartment at +37°C for 4 h. Cultures were then washed again twice to remove all agents.
  • PMNs or THP-1 cells were preincubated with the peptide inhibitor or antibody studied for 1 h before transfer to the upper compartment (lxlO 5 cells in lOO ⁇ l RPMI/0.1 % BSA or the complete 10 % FCS-containing medium).
  • PMNs were allowed to migrate for 2 h through the LLG-C4-GST coated membrane and for 30 min through the HMEC monolayer.
  • THP-1 cells were allowed to migrate for 16 h. The non-migrated cells were removed from the upper surface by a cotton swab and the cells that had traversed the filters were stained with crystal violet and counted.
  • mice at the age of 31-32 weeks were injected intraperitoneally with 3% (w/v) thioglycolate in sterile saline (36).
  • Peptides (5-500 ⁇ g in lOO ⁇ l) were introduced intravenously through the tail vein.
  • Animals were euthanized after 3 h and the peritoneal cells were harvested by injecting 10 ml of sterile PBS through the peritoneal wall. Red blood cells present in the lavage fluid were removed by hypotonic lysis. Cells were centrifuged and resuspended in 1 ml of sterile 0.25% BSA/Krebs-Ringer. The supernatants were also collected and analysed by gelatin zymography.
  • the number of neutrophils was determined following staining with 0.1% crystal violet and using a light microscope equipped with a x 1 00 objective. F or immunofluorescence staining, c ells were allowed to bind to poly-L-lysine coated cover slips, fixed with 2.5% paraformaldehyde in PBS at +4°C for 30 min followed by several w ashings. The Fc receptors were blocked in the presence of 20% of rabbit serum and 3% BSA in PBS. The cells were then incubated with anti-MMP-9 polyclonal and U M monoclonal (MCA74) antibodies for 30 min.
  • MCA74 monoclonal
  • the secondary antibodies After washing with PBS, the secondary antibodies, rhodamine (TRITC)-conjugated anti-rabbit or FITC-conjugated anti-rat (Fab') 2 were incubated for another 30 min. The samples were examined with a confocal microscope. The animal studies were approved by an ethical committee of Helsinki University.
  • phage peptide display libraries we selected peptides that interact with the ⁇ M I domain.
  • GST-binding phage were first eliminated on GST-coated wells and the unbound phage preparations were incubated on ⁇ M I domain GST fusion protein-coated wells.
  • the ⁇ M I domain-binding phage were enriched by three rounds of panning and the peptide sequences were determined. With the exception of one linear peptide, the peptides were derived from the cyclic CX 7 C and CX 8 C libraries.
  • the I domain-binding sequences showed only one conserved motif, a somewhat unexpected finding in terms of the known ligand binding promiscuity of the I domain.
  • the bound peptides contained two consecutive negatively charged amino acids, i.e. glutamatic and/or aspartatic acids, followed by glycine and tryptophan residues (Fig. 1A).
  • the consensus D/E-/D/E-G/L- W determined by this approach was clearly different from LLG-C4 and other ⁇ 2 integrin-binding peptides reported so far.
  • phage display peptides as intein fusion proteins, from which the peptides were cleaved. This allowed us to rapidly test the peptide solubility and the binding specificity before large-scale chemical peptide synthesis.
  • the peptides were cloned using oligonucleotide primers that amplify the peptide library insert from the phage vector. Consequently, all the peptides prepared contain the vector-derived sequences ADGA and GAAG in the NH 2 - and COOH- termini, respectively.
  • ADGA-CILWMDDGWC-GAAG p eptide ADGA-CILWMDDGWC-GAAG
  • DDGW p eptide ADGA-CILWMDDGWC-GAAG
  • the DDGW-bearing phage also showed also specific binding to the ⁇ L I domain that was inhibitable by DDGW but the interaction was weaker than with the ⁇ M I domain (Fig. IC and data not shown). No binding was observed with the ⁇ x I domain or GST used as a control (Fig. IC).
  • CPEELWWLC phage library-derived peptides
  • DELW(S/T)LG sequence present on the catalytic domain of MMP-2 and MMP-9 gelatinases (Fig. 1A).
  • DELW-like sequences with double negative charges are also present in other secreted MMPs but not in the membrane-type MMPs such as MMP- 14.
  • MMP-9 in particular could be a ligand of the ⁇ integrins as MMP-9 gelatinase is the major leukocyte MMP and is induced during ⁇ 2 integrin activation.
  • MMP-9 in particular could be a ligand of the ⁇ integrins as MMP-9 gelatinase is the major leukocyte MMP and is induced during ⁇ 2 integrin activation.
  • a first step we synthesized the whole proMMP-9 sequence as overlapping 20-mer peptides on a pepspot membrane. Binding assays with the ⁇ M I domain revealed a single active peptide that located to the MMP-9 catalytic domain (Fig. ID). No binding was observed, when the I domain was omitted and the membrane was probed with antibodies only.
  • the sequence of the I domain-binding peptide was QGDAHFDDDELWSLGKGVVV and it contained the binding motif identified by phage display (Fig. ID).
  • the active MMP-9 peptide contained four consecutive amino acids with negative charges, DDDE. To study the importance of these residues, the aspartic and glutamic acid residues that were closest to the tryptophan were replaced by alanines. At the same time the peptide length was shortened to 15-mer. The alanine mutagenesis significantly abrogated I domain binding on the pepspot filter; the OD value dropped from 2010 to 476 (Table I). To study whether the negatively charged peptide from other MMPs is also active, we synthesized the corresponding 15-mers and the double alanine mutations.
  • CEDGWC-GAAG but not ADGA-CDDGWC-GAAG was the minimal peptide that supported ⁇ M I domain binding.
  • the longer side chain of glutamate compared to aspartate is probably required to bring the negatively charged carboxyl group in the correct position for I domain binding.
  • Progelatinases bind to purified ⁇ jv ⁇ 2 and ⁇ ⁇ 2 integrins and their I domains
  • the DDGW peptide was an efficient inhibitor and it inhibited proMMP-9 binding to the ⁇ M I domain with an IC50 of 20 ⁇ M (Fig. 3 A and 3B).
  • the peptide ADGACILWMKKGWCGAAG (KKGW) containing lysines in place of aspartic acids was prepared.
  • the KKWG peptide was inactive and did not compete with proMMP-9 binding.
  • lovastatin as its binding site in the ⁇ L I domain is known (34, 35). Lovastatin was not able to compete with proMMP-9 even at a high concentration.
  • EDTA cation chelator
  • a non-peptide chemical MMP inhibitor (Inhl) also prevented proMMP-9 binding.
  • integrin blocking antibodies and ligand peptides As EDTA inhibits both the gelatinase and the integrin, we used integrin blocking antibodies and ligand peptides to demonstrate the specific binding activity of ⁇ 2 integrin.
  • the known ligand-binding blocking antibodies MEM 170, MEM 83, and LM2/1 inhibited proMMP-9 binding.
  • a control antibody TL3 had no effect.
  • the I domain binding peptide LLG-C4 showed a partial inhibitory effect.
  • RGD-4C a ligand of ⁇ v integrins, served as control peptide and had no effect on proMMP-9 binding.
  • the purity of the integrins was typically more than 90% and that of I domains 95%, making it unlikely that progelatinases would bind to impurities in the preparations.
  • Progelatinase-integrin complexes were also obtained by co-precipitation experiments using HT1080 conditioned medium as a source of proMMP-9 and proMMP-2, which were analyzed by zymography.
  • the progelatinases co-precipitated with M ⁇ 2 integrin or ⁇ M I domain GST fusion protein when these were used as a bait.
  • the integrin added to the medium was immunoprecipitated with the ⁇ M antibody OKM10 (Fig. 4A).
  • the ⁇ M I domain GST protein was pulled down with glutathione-beads (Fig. 4B). CTT but not STT had an inhibitory effect.
  • CTT and Inhl prevented the binding of proMMP-9 to the integrin
  • CTT and Inhl avidly bind to proMMP-9.
  • ProMMP-9 specifically bound to the CTT-GST fusion protein (Fig. 5A) but not to LLG-C4-GST.
  • CTT and Inhl at 100 ⁇ M concentrations effectively competed in binding but W ⁇ A CTT did not.
  • the proMMP-9 preparation did not contain detectable amounts of active MMP-9 on zymography analysis, and after proMMP-9 activation with APMA, the CTT-GST binding increased.
  • CTT and Inhl could also bind to proMMP-9 secreted into the medium of PDBu-activated THP-1 leukemic cells (Fig. 5B) or HT1080 fibrosarcoma cells (not shown).
  • a time-dependent reduction in the gelatinolytic activity of proMMP-9 was observed with CTT (panel 1) and Inhl (panel 3), but not with the W ⁇ A CTT peptide (panel 4).
  • the CTT complex was reversible and disappeared after repeated freezing and thawing of the samples.
  • THP-1 monocytic leukemia cells in the resting state and after induction by PDBu, which mimics leukocyte activation in vivo.
  • THP-1 cell-stimulation with PDBu led to upregulation of MMP-9 (data not shown).
  • the cell surface glycoproteins of THP- 1 cells were labelled with tritium [ H] followed by immunoprecipitation with ⁇ 2 integrin and MMP-9 antibodies.
  • the ⁇ M chain antibody OKM10 and ⁇ 2 chain antibody 7E4 immunoprecipitated two [ 3 H] -labelled proteins corresponding to the integrin ⁇ chain (165 kDa) and ⁇ 2 chain (95 kDa) (Fig. 6A, lanes 9-10).
  • polyclonal MMP-9 antibodies immunoprecipitated the same two integrin chains (lane 7).
  • essentially no co-precipitation of ⁇ M and ⁇ 2 were observed with MMP-9 antibodies, although the ⁇ M and ⁇ 2 chains were present.
  • the co- precipitation o f t he i ntegrin c hains b y M MP-9 a ntibodies w as p revented b y t he C TT peptide (lane 8).
  • the control antibody (TL3) did not precipitate any proteins.
  • THP-1 cells do not express high amounts of the ⁇ L chain (20)
  • the Jurkat T cell line which expresses more ⁇ L than ⁇ M (28).
  • the ⁇ L antibody co-precipitated more proMMP-9 in comparison to the ⁇ M antibody.
  • ProMMP-9 and ⁇ M ⁇ 2 were found to co-localize on the cell surface following PDBu- activation of THP-1 cells as studied by fluorescence and confocal microscopy (Figs. 7A and 7B, respectively). Using a higher magnification, colocalization was primarily seen in cell surface clusters (Fig. 7B), and to a lesser extend on areas where cells contacted each other (not shown).
  • DDGW peptide is an integrin ligand
  • the recombinant intein-produced ADGA-CPCFLLGCC-GAAG peptide supported adhesion, but unlike the DDGW peptide, it also supported adhesion in the absence of integrin activation.
  • the acute myeloid leukemic cell line OCI/AML-3 also avidly adhered to DDGW, whereas human fibrosarcoma HT1080 cells which lack ⁇ integrins did not (not shown).
  • THP-1 cells were able to adhere on DDGW, we next studied the effect of the peptide on ⁇ 2 integrin dependent adhesion to fibrinogen and ICAM-1.
  • DDGW did not block cell adhesion to fibrinogen
  • the LLG-C4 peptide blocked the adhesion as previously reported (Fig. 8B).
  • DDGW did not block the binding of recombinant ⁇ M I domain to immobilized fibrinogen (not shown).
  • DDGW did not either block cell adhesion on ICAM-1 -Fc fusion protein.
  • the blocking antibody 7E4 against ⁇ 2 integrins prevented the ICAM-1 binding, indicating that the THP-1 cells bound in a ⁇ 2 integrin dependent manner (Fig. 8C).
  • Fig. 8C We also found no blocking effect of DDGW on THP-1 adhesion to LLG-C4-GST fusion protein (not shown).
  • THP-1 cells were cultured for 48 h in the presence of DDGW, an increase of proMMP-9 level was observed in the conditioned medium as studied by gelatin zymography (Fig. 8D).
  • the peptide increased both monomeric and dimeric proMMP-9 in the culture medium.
  • CTT slightly decreased or inhibited active proMMP-9.
  • KKGW and W ⁇ A CTT had no effect.
  • the soluble LLG-C4 peptide also blocked the migration. In the presence of GST coating, cell migration was neglible. To verify that the effect of DDGW peptide was ⁇ 2 integrin dependent, HT1080 fibrosarcoma cells lacking these integrins were allowed to migrate in the presence of CTT, DDGW, KKGW or LLG-C4. Of these peptides, only CTT was capable of inhibiting cell migration (Fig. 9C).
  • Peptide inhibitors of the proMMP-9/ ⁇ M ⁇ 2 complex prevent neutrophil migration
  • pepspot analysis located the integrin interactive site of proMMP-9 to a 20-amino acid long sequence present in the catalytic domain, QGDAHFDDDE- LWSLGKGVVV. Further screening by the pepspot system has indicated that sufficient integrin binding activity is achieved by truncating this sequence to a hexapeptide, HFDDDE (data not shown).
  • a short sequence is the bioactive site of proMMP-9 (Fig. 10A).
  • ⁇ MMP-9 is composed of the prodomain (Pro) and the catalytic domain but lacks the hemopexin domain.
  • the fibronectin type II repeats (Fnll) were also produced as a separate recombinant protein as this is an important substrate-binding region.
  • the procatalytic domain construct ⁇ MMP-9 bound the ⁇ M I domain nearly as efficiently as the wild type proMMP-9 (Fig. 10B). Fnll protein almost lacked activity.
  • the HFDDDE peptide identified by the solid-phase pepspot analysis was highly active when made by peptide synthesis and inhibited proMMP-9 binding to the ⁇ M I domain with an IC 50 of 20 ⁇ M (Fig. 10C).
  • the bound proMMP-9 was determined with the GE-213 antibody, which recognizes an epitope of the Fnll domain (data not shown).
  • a scrambled peptide DFEDHD with the same set of negatively charged amino acids was inactive.
  • HFDDDE was equally potent as DDGW, the ⁇ M I domain-binding peptide discovered by phage display and described above.
  • KKGW the control peptide for DDGW, was without effect.
  • As the HFDDDE sequence is highly conserved in the members of the MMP family, we also examined the ⁇ I domain binding to human neutrophil collagenase, MMP-8. I domain showed a similar DDGW-inhibitable binding to proMMP-8 as to proMMP-9 (Fig. 10D). ICAM-1 and fibrinogen did not compete with either proMMP, implying different binding sites for the matrix proteins and proMMPs in the I domain.
  • PMNs After integrin activation, PMNs exhibited an ability to adhere on proMMP-9. PMA- stimulated PMNs bound to microtiter well-coated ⁇ MMP-9 nearly as strongly as to proMMP-9 (Fig. 11 A). Stimulation of PMNs with C5a or TNF- ⁇ gave similar results PMN adherence increasing by 3-fold (Fig. 1 IB). The Fnll domain did not support PMN adhesion. PMN adherence was inhibited by HFDDDE (50 ⁇ M), DDGW (50 ⁇ M), the soluble ⁇ M I domain and the MEM 170 antibody (Fig. 11C), indicating ⁇ 2 integrin- directed binding.
  • the control peptides (DFEDHD, KKGW) and an irrelevant monoclonal antibody (anti-GPA) had no effect.
  • the CTT peptide but not the W ⁇ A CTT control peptide lacking MMP inhibitory activity, binds to the MMP-9 catalytic domain (unpublished results) and also inhibited the PMN adherence. MMP-9 antibodies inhibited partially.
  • ⁇ M ⁇ 2 -transfected L cells We also examined ⁇ M ⁇ 2 -transfected L cells.
  • the ⁇ M ⁇ 2 L-cell transfectants bound to proMMP-9 and ⁇ MMP-9 similarly as PMNs did and the I domain ligands and MMP-9 inhibitors attenuated the binding (Fig. 1 ID).
  • the transfected cells also showed a weak adherence to Fnll domain, but the studied peptides and antibodies did not inhibit this binding. Wild type L cells or LAD-1 cells showed no binding to proMMP-9 or its domains.
  • DDGW peptide can release proMMP-9 from THP-1 cells.
  • HFDDDE peptide also released proMMP-9 but was less effective than DDGW (Fig. 12D).
  • the scrambled peptide did not induce the release of proMMP-9.
  • DDGW concentration-dependent and up to 90 % inhibition w as o btained b y doses of 5 O ⁇ g and 5 OO ⁇ g er mouse, respectively.
  • DDGW was active even at 5 ⁇ g given per mouse corresponding to an effective dose of 0.1 mg/kg mouse tissue.
  • PMNs were present intraperitoneally after thioglycolate-stimulus in comparison to the PBS control.
  • the collected inflammatory PMNs stained positively for the proMMP-9/ ⁇ M ⁇ 2 complex by double immunofluorescence (Fig. 13B).
  • the cells collected after PBS injection lacked the complex; they expressed the integrin but had no cell-surface MMP-9 (Fig. 13C).

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GB2429012A (en) * 2005-08-12 2007-02-14 Cartela Ab Polypeptides capable of binding an integrin I-domain.
WO2007020405A2 (en) * 2005-08-12 2007-02-22 Cartela R & D Ab Integrin i-domain binding peptides
JP5749651B2 (ja) 2008-11-04 2015-07-15 インデックス・ファーマシューティカルズ・アクチエボラーグ 多形核細胞の動員および／または遊走を減少させる化合物および方法
US8003110B1 (en) * 2011-01-24 2011-08-23 Matthias W. Rath Metalloproteinase oligopeptides and their therapeutic use
JP2020511517A (ja) * 2017-03-22 2020-04-16 ザ チルドレンズ メディカル センター コーポレーション Ｐｒｓｓ２阻害
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