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WO2000032759A1 - Activateur de plasminogene humain - Google Patents

Activateur de plasminogene humain Download PDF

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Publication number
WO2000032759A1
WO2000032759A1 PCT/US1999/009991 US9909991W WO0032759A1 WO 2000032759 A1 WO2000032759 A1 WO 2000032759A1 US 9909991 W US9909991 W US 9909991W WO 0032759 A1 WO0032759 A1 WO 0032759A1
Authority
WO
WIPO (PCT)
Prior art keywords
plasminogen
asp
sequence
polypeptide
seq
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.)
Ceased
Application number
PCT/US1999/009991
Other languages
English (en)
Inventor
Xinli Lin
Xuejun C. Zhang
Jordan J. N. Tang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Oklahoma Medical Research Foundation
Original Assignee
Oklahoma Medical Research Foundation
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Oklahoma Medical Research Foundation filed Critical Oklahoma Medical Research Foundation
Priority to EP99921757A priority Critical patent/EP1135475A1/fr
Priority to CA002353699A priority patent/CA2353699A1/fr
Priority to AU38880/99A priority patent/AU3888099A/en
Publication of WO2000032759A1 publication Critical patent/WO2000032759A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • 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
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
    • 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
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6456—Plasminogen activators
    • C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21069—Protein C activated (3.4.21.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • human plasmin does not have a similar six-residue loop and can not activate plasminogen.
  • the position of the peptide in the other proteins corresponds to a position in plasminogen of between Arg- 644 and Lys-645 of human plasminogen.
  • Recombinant D A technology was used to insert the loop sequence Asp-Asp-Asp-Thr-Tyr-Asp (amino acids 399- 404 in SEQ ID NO:2) in micro-plasminogen between residues 644 and 645 (using the full plasminogen numbering).
  • micro-plasminogen [+6] The modified n ⁇ cro-plasminogen (called micro-plasminogen [+6]) was activated by urokinase to micro-plasmin[+6], which was tested for its ability to activate human rmcro-plasminogen to micro-plasmin.
  • the protein may be made by chemically modifying a native protein Generally, this requires inserting the recognition/ activation peptide into the native protein This may be accomplished chemically or enzymatically using, for example, a peptidase
  • the proteins will be synthesized using recombinant methodology Generally, this involves creating a polynucleotide sequence that encodes the protein, placing the polynucleotide in an expression cassette under the control of a suitable expression promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein
  • DNA encoding a protein can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al., Meth. Enzymol. 1979; 68: 90-99; the phosphodiester method of Brown et al., Meth. Enzymol.
  • partial length sequences may be cloned and the appropriate partial length sequences cleaved using appropriate restriction enzymes. The fragments may then be ligated to produce the desired DNA sequence.
  • DNA encoding the protein will be produced using DNA amplification methods, for example polymerase chain reaction (PCR).
  • the proteins may be expressed in a variety of host cells, preferably E. coli or other bacterial hosts. Plasmids encoding the protein can be transferred into the chosen host cell by well-known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for yeast or mammalian cells. Cells transformed with the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.
  • proteins should be assayed for biological activity.
  • assays well known to those of skill in the art, generally fall into two categories; those that measure the binding affinity of the protein to a particular target, and those that measure the biological activity of the protein.
  • Plasminogen activators including the plasminogen recognition/ activation peptide can be administered for treatment of blood clot disorders, such as in heart attacks, as known in the art for administration of native streptokinase and tissue-type plasminogen activator (t-PA) and urokinase (u- PA or UK).
  • t-PA tissue-type plasminogen activator
  • u- PA urokinase
  • the compounds are preferably administered intravenously in appropriate carriers.
  • the appropriate dosages will depend upon the route of administration and the treatment indicated, and can be readily determined by one skilled in the art. Dosages are generally initiated at lower levels and increased until desired effects are achieved.
  • DDDTYD amino acids 399-404 of SEQ ID NO: 2
  • pETl 1- Pg 6 amino acid sequence between R644 and K645 (Pig sequence number). This was done by modifying the / Pg expression plasmid, pETl 1- Pg, using a recombination PCR method. Two PCR (PCR-1 and PCR- 2) were performed first using pETl 1- ⁇ Pg as template and primers P1/P2 and P3/P4
  • the resulting PCR product which is about 1.1 Kb, was cut with Stu I/Hind HI, and the 0.86 Kb fragment was cloned into the Stu I/Hnd HI cut of vector pET 11 - ⁇ Pg.
  • the resulting plasmid pET 11 - ⁇ Pg-D6 was transformed into E. coli BL21(DE3), and protein was expressed as inclusion bodies, which were purified using standard procedures.
  • the purified inclusion bodies were dissolved in 8 M urea, and the protein was refolded by dialysis.
  • the refolded protein was partially purified using Sephacryl S-300 gel filtration and FPLC Resource Q.
  • Example 2 Properties of the recombinant micro-plasminogen 1+61 Plasmin activity was measured.
  • Micro-plasminogen (substrate) was incubated with SK in buffer A at 37 °C, to activate the micro-plasminogen before the chromogenic substrate was added. The reaction was continued for 5 to 10 min, and then was stopped by adding 0.2 M acetic acid. Reactions were performed in 96-well microtiter plates and absorption at 405 nm was recorded. The results are shown in Table I, where ⁇ Pg[+6] is micro-plasminogen[+6] and ⁇ Pg is micro-plasminogen. Table I. The activity of micro-plasminogen [+6]
  • micro-plasminogen[+6] has no activity (Table I, line 1) and micro-plasminogen[+6] can not activate plasminogen (Table I, line 2 as compared to Table I, line 3).
  • Micro-plasminogen[+6] can not be activated by SK (Table I, line 4).
  • the complex or activated micro-plasmin[+6] can activate plasminogen (Table 1-6 as compared to Table I, line 5).
  • micro-plasminogen[+6] are different from those of micro-plasminogen.
  • the activity of plasminogen activation shown in Table I, line 6, can come either from micro-plasmin[+6] or from its complexation with streptokinase.
  • Table H shows the plasminogen activation activity of UK at different dilutions.
  • Table 13, line 2 shows the activity of the urokinase- micro- plasminogen [+6] complex, which was pre-incubated to convert micro- plasminogen[+6] to micro-plasmin[+6] prior to the addition of the micro- plasminogen substrate, at the same dilutions as in line 1.
  • the plasmin activity of complex was twice that of urokinase at all dilutions.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Cette invention concerne des protéines qui jouent un rôle dans la formation de complexes ou l'activation de plasminogène. Des parties comprennent un peptide à six acides aminés qui peut être utilisé en qualité d'activateur de plasminogène. Ce peptide est particulièrement utile lorsqu'il est inséré dans un plasminogène ou une séquence de plasmine, notamment lorsqu'il est inséré entre les résidus d'acide aminé correspondant aux résidus d'acide aminé 644 et 645 d'un plasminogène humain dans toute sa longueur.
PCT/US1999/009991 1998-12-02 1999-05-06 Activateur de plasminogene humain Ceased WO2000032759A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP99921757A EP1135475A1 (fr) 1998-12-02 1999-05-06 Activateur de plasminogene humain
CA002353699A CA2353699A1 (fr) 1998-12-02 1999-05-06 Activateur de plasminogene humain
AU38880/99A AU3888099A (en) 1998-12-02 1999-05-06 Human plasminogen activator

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US11058898P 1998-12-02 1998-12-02
US60/110,588 1998-12-02

Publications (1)

Publication Number Publication Date
WO2000032759A1 true WO2000032759A1 (fr) 2000-06-08

Family

ID=22333848

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/009991 Ceased WO2000032759A1 (fr) 1998-12-02 1999-05-06 Activateur de plasminogene humain

Country Status (4)

Country Link
EP (1) EP1135475A1 (fr)
AU (1) AU3888099A (fr)
CA (1) CA2353699A1 (fr)
WO (1) WO2000032759A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009116035A1 (fr) * 2008-03-16 2009-09-24 Hadasit Medical Research Services & Development Ltd. Mutant du tpa utilisé dans le traitement des lésions cérébrales aiguës et des affections neurodégénératives

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019793A2 (fr) * 1990-06-14 1991-12-26 Beecham Group Plc Activateurs de plasminogene hybrides
WO1992004450A1 (fr) * 1990-09-01 1992-03-19 Beecham Group Plc Activateurs de plasminogene hybrides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991019793A2 (fr) * 1990-06-14 1991-12-26 Beecham Group Plc Activateurs de plasminogene hybrides
WO1992004450A1 (fr) * 1990-09-01 1992-03-19 Beecham Group Plc Activateurs de plasminogene hybrides

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WANG E.A.: "Crystal structure of the catalytic domain of the human plasmin complexed with streptokinase", SCIENCE, vol. 281, 11 September 1998 (1998-09-11), LANCASTER, PA US, pages 1662 - 1665, XP002113671 *
YOUNG E.A.: "Plasminogen activation by streptokinase via a unique mechanism", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 273, no. 5, 30 January 1998 (1998-01-30), MD US, pages 3110 - 3116, XP002113670 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009116035A1 (fr) * 2008-03-16 2009-09-24 Hadasit Medical Research Services & Development Ltd. Mutant du tpa utilisé dans le traitement des lésions cérébrales aiguës et des affections neurodégénératives
US9072737B2 (en) 2008-03-16 2015-07-07 Hadasit Medical Research Services & Development Ltd. tPA mutant in the treatment of acute brain injury and neurodegenerative disorders

Also Published As

Publication number Publication date
CA2353699A1 (fr) 2000-06-08
AU3888099A (en) 2000-06-19
EP1135475A1 (fr) 2001-09-26

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