WO2005081622A1 - Procedes et compositions de detection de l'oxyde nitrique - Google Patents

Procedes et compositions de detection de l'oxyde nitrique Download PDF

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Publication number
WO2005081622A1
WO2005081622A1 PCT/IB2005/000301 IB2005000301W WO2005081622A1 WO 2005081622 A1 WO2005081622 A1 WO 2005081622A1 IB 2005000301 W IB2005000301 W IB 2005000301W WO 2005081622 A1 WO2005081622 A1 WO 2005081622A1
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WO
WIPO (PCT)
Prior art keywords
thiyl radical
reacting compound
marker
dmpo
thiyl
Prior art date
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PCT/IB2005/000301
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English (en)
Inventor
B. Mason Hughes
Christine Kornmeier
Thomas Misko
Barnett Pitzele
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Pharmacia & Upjohn Company Llc
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.)
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Application filed by Pharmacia & Upjohn Company Llc filed Critical Pharmacia & Upjohn Company Llc
Publication of WO2005081622A1 publication Critical patent/WO2005081622A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6806—Determination of free amino acids
    • G01N33/6812—Assays for specific amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials

Definitions

  • NO nitric oxide
  • S-nitrosation or S-nitrosylation a chemical modification of cysteine residues
  • the adduct of cysteine is termed a nitrosothiol and accounts for the NO-dependent alteration of the activity of proteins including H-ras, the olfactory cyclic nucleotide- gated channel, and glyceraldehyde-3 -phosphate dehydrogenase (GAPDH).
  • Nitrosothiols are frequently labile because of their reactivity with intracellular reducing agents, such as ascorbic acid and glutathione (GSH), and with reduced metal ions, especially Cu(I). This lability may result in tissue half-lives of seconds to a few minutes.
  • WO 2002/0391 19 Al describes a method for detecting S-nitrosothiols wherein a test sample comprising at least one protein substrate is treated with an alkylthiolating agent to block free thiol groups on the protein substrate, nitrosothiol bonds on the protein substrate are reduced to form free thiol groups, the alkylthiolating agent is removed from the test sample, free thiol groups on the protein substrate are reacted with a detectably tagged, activated mixed disulfide, transferring the detectable tag to the protein and the detectable tag on the protein substrate is detected.
  • DMPO dimethyl- 1- ⁇ yrroline N-oxide
  • PBN phenyl-N-t-butylnitrone
  • BMPO methyl-l-pyrroline N-oxide
  • EPR Electron Paramagnetic Resonance
  • S-nitrosylated proteins and peptides can be photolytically decomposed in the presence of DMPO (PBN, BMPO) thus forming specific and stable protein/peptide-thiyl-DMPO (PBN, BMPO) adducts.
  • DMPO DMPO
  • PBN, BMPO protein/peptide-thiyl-DMPO
  • S-nitroso-proteins and peptide adducts can be identified in mass spectrometry by their mass shift, and exact neutral loss.
  • the present invention provides a method of selectively and specifically labeling, purifying, and/or identifying S-nitrosothiols and all types of labeling or modifications which may be made to DMPO, PBN, BMPO, or other thiyl radical reactive compounds, that can be used to selectively label S-nitrosothiols.
  • Labeling and modifications can include biotinylation, his-tagging, l4 C or other radiolabel, and fluorescent tagging. Biotinylation, his-tagging, or other methods of labeling DMPO, PBN, BMPO, or other thiyl reactive compounds, allows for the immunochemical purification, concentration, and visualization of these adducted S-nitrosylated proteins or peptides. Otherwise undetectable trace quantities of nitrosylated proteins are purified for identification from biological fluids, tissue lysates, tissue sections, and tissue culture cell supernatants and lysates.
  • fluorescent tagging or radiolabelling (such as, for example l4 C and the like) of DMPO, PBN, BMPO, or other thiyl radical reactive compounds, provides for the detection, visualization, and localization of S-nitrosylated proteins, peptides, and compounds in biological fluids, tissue lysate, tissue sections, and tissue culture cells with or without supernatants.
  • Antibodies to DMPO may also be used to detect S- nitrosylated proteins in the practice of the present invention.
  • DMPO means 2H-Pyrrole, 3,4-dihydro-2,2- dimethyl-, 1 -oxide (9CI) , CAS Registry number 3317-61 -1.
  • DMPO has the following structure, wherein Me represents methyl: o Me
  • DMPO is also sometimes known as 1-Pyrroline, 5,5-dimethyl-, 1 -oxide (6CI, 7CI, 8CI); 2,2-Dimethyl-3,4-dihydro-2H-pyrrole N-oxide; 5,5-Dimethyl- ⁇ 1-pyrroline 1 - oxide; 5, 5-Dimethyl- ⁇ 1-pyrroline N-oxide; 5,5-Dimethyl- 1 -pyrroline 1 -oxide; 5,5- Dimethyl- 1 -pyrroline N-oxide; and 5,5-Dimethyl-4,5-dihydro-3H-pyrrole N-oxide.
  • BMPO means 2H-Pyrrole-2-carboxylic acid, 3,4- dihydro-2-methyl-, 1,1-dimethylethyl ester, 1 -oxide (9CI) , CAS Registry number 387334-31-8.
  • BMPO has the following structure, wherein Me represents methyl, and Bu-t represent tert-butoxycarbonyl: o I I Me N. ' ⁇ C — OBu-t
  • BMPO is also sometimes known as BocMPO.
  • PBN is also sometimes known as Nitrone, N-tert-butyl- ⁇ -phenyl- (6CI, 7CI, 8CI); ⁇ - Phenyl-N-tert-butylnitrone; ⁇ -Phenyl-tertbutyl nitrone; 2-Methyl-N- (phenylmethylene)-2-propanamine N-oxide; 2-Phenyl-N-tert-butylnitrone; Benzylidene-tert-butylamine N-oxide; Benzylidene-tert-butylamine oxide; C-Phenyl- N-tert-butylnitrone; C-Phenyl-N-ter/-butylnitrone; N-Benzylidene-tert-butylamine N- oxide; N-Benzylidene-tert-butylamine oxide; N-tert-Butyl- ⁇ -phenylnitrone; N-tert- Butyl-2-phen
  • POBN means 2-Propanamine, 2-methyl-N-[(l- oxido-4-pyridinyl)methylene]-, N-oxide (9CI) , CAS registry number 66893-81-0. POBN has the structure:
  • Biotin as used herein, means lH-Thieno[3,4-d]imidazole-4- pentanoic acid, hexahydro-2-oxo-, (3aS,4S,6aR)- (9CI) , CAS Registry number 58-85- 5. Biotin has the structure:
  • Biotin is also sometimes known as lH-Thieno[3,4-d]imidazole-4-pentanoic acid, hexahydro-2-oxo-, [3aS-(3a ⁇ ,4 ⁇ ,6a ⁇ )]-; (+)-Biotin; (+)-cis-Hexahydro-2-oxo-lH- thieno[3,4]imidazole-4-valeric acid; Biodermatin; Bioepiderm; Bios II; cis-(+)- Tetrahydro-2-oxothieno[3,4]imidazoline-4-valeric acid; Coenzyme R; D(+)-Biotin; D- Biotin; d-Biotin; Factor S; Factor S (vitamin); Lutavit H2; Meribin; Rovimix H 2; Vitamin B7; and Vitamin H.
  • his-tag and "hexahistidine,” as used herein, means a peptide fragment comprising six or more consecutive histidine (his) residues in a row that will act as a metal binding site, and can be added to a thiyl-reacting compound. The resulting his-tag may be isolated by metal chelate affinity chromatography, for example, and purified from solution.
  • hv represents radiation energy, h is Planck's constant (equal to 6.626 x 10 "27 erg-seconds, or 6.626196 x 10 "34 J s), and v is frequency. Particularly useful radiation energy in the practice of the present invention is green light in the visible spectrum.
  • ultraviolet radiation may be used, wherein the wavelength ( ⁇ ) is between 380 and 3000 nm (nanometers), and the frequency (v) is between 7.9 x 10 and lx 10 16 Hz (hertz, or cycles per second).
  • Detecting devices include, without limitation, spectrophotometers, including UV spectrophotometers, mass spectrometers, scintillation counters, and the like. Those skilled in the art will appreciate that under appropriate conditions, that is, using appropriate markers, an appropriate detecting device matched with the marker may be selected. For example, where a marker comprises a radioactive isotope, a scintillation counter may be employed.
  • a labeled thiyl radical-reactive compound useful in the present invention is contacted with a thiyl radical from an irradiated S-nitrosylated protein or peptide and the light generated thiyl radical formed from the S-nitrosylated protein or peptide is detected through the label.
  • the thiyl radical -reactive agent DMPO is labeled with biotin.
  • the biotin molecule is spatially separated from the DMPO molecule by a spacer.
  • the spacer operably links the DMPO molecule with the biotin molecule.
  • the spacer may be, for example, a hydrocarbon, such as an alkylene group.
  • the spacer is a Ci - C 8 alkylene group.
  • An S- nitrosylated peptide for example, S-nitrosylated glutathione (GS) is exposed to radiation, such as visible green light radiation.
  • the visible light spectrum radiation ionizes the S-nitrosylated GS, freeing a nitric oxide radical and forming a thiyl radical at the locus of the previously nitrosylated cysteine of GS.
  • a labeled thiyl radical- reactive compound such as biotinylated DMPO, for example, is contacted with the thiyl radical of the GS, and forms a stable adduct with the GS.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Biotechnology (AREA)
  • Pathology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de détection de protéines ou de peptides S-nitrosylatés dans un échantillon biologique. Ce procédé consiste à: soumettre d'abord les S-nitrosothiols à un clivage photolytique dans les protéines et les peptides S-nitrosylés pour former des groupes à radical thiyl; mettre ensuite l'échantillon biologique en contact avec un composé de réaction avec le radical thiyl, ce dernier étant modifié avec au moins un marqueur; et enfin, détecter ledit marqueur avec un dispositif de détection approprié.
PCT/IB2005/000301 2004-02-17 2005-02-07 Procedes et compositions de detection de l'oxyde nitrique WO2005081622A1 (fr)

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US54502404P 2004-02-17 2004-02-17
US60/545,024 2004-02-17

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009088801A1 (fr) * 2007-12-31 2009-07-16 Medical College Of Wisconsin Composés de piégeage de spin à base de nitrone bifonctionnel et trifonctionnel, et leurs utilisations
CN101893634A (zh) * 2009-05-20 2010-11-24 中国科学院生物物理研究所 特异检测蛋白质或多肽半胱氨酸巯基修饰的方法及其用途

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994002865A1 (fr) * 1992-07-23 1994-02-03 The United States Of America, Represented By The Secretary, Department Of Health And Human Services Detection de l'oxyde nitrique par resonance de spin electronique
US5459076A (en) * 1992-04-22 1995-10-17 Brigham And Women's Hospital Method for detecting nitric oxide, nitrosonium equivalents, S-nitrosothiols and S-nitroso-proteins in biological systems
WO2002016934A1 (fr) * 2000-08-25 2002-02-28 Queen Mary & Westfield College Essais relatifs a des composes s-nitrosothiol
WO2002039119A2 (fr) * 2000-10-27 2002-05-16 The Johns Hopkins University Recherche de nytrosilation des protéines
WO2004002536A1 (fr) * 2002-06-28 2004-01-08 Pharmacia Corporation Agents de contraste des plus utiles pour quantifier de l'oxyde nitrique et methodes a cet effet
US20040203068A1 (en) * 2001-12-12 2004-10-14 Joan Mannick System for detection of nitrosylated proteins

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5459076A (en) * 1992-04-22 1995-10-17 Brigham And Women's Hospital Method for detecting nitric oxide, nitrosonium equivalents, S-nitrosothiols and S-nitroso-proteins in biological systems
WO1994002865A1 (fr) * 1992-07-23 1994-02-03 The United States Of America, Represented By The Secretary, Department Of Health And Human Services Detection de l'oxyde nitrique par resonance de spin electronique
WO2002016934A1 (fr) * 2000-08-25 2002-02-28 Queen Mary & Westfield College Essais relatifs a des composes s-nitrosothiol
WO2002039119A2 (fr) * 2000-10-27 2002-05-16 The Johns Hopkins University Recherche de nytrosilation des protéines
US20040203068A1 (en) * 2001-12-12 2004-10-14 Joan Mannick System for detection of nitrosylated proteins
WO2004002536A1 (fr) * 2002-06-28 2004-01-08 Pharmacia Corporation Agents de contraste des plus utiles pour quantifier de l'oxyde nitrique et methodes a cet effet

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BERLINER L J ET AL: "UNIQUE IN VIVO APPLICATIONS OF SPIN TRAPS", FREE RADICAL BIOLOGY AND MEDICINE, ELSEVIER SCIENCE, vol. 30, no. 5, 1 March 2001 (2001-03-01), pages 489 - 499, XP001155626, ISSN: 0891-5849 *
ROSSI R ET AL: "A method to study kinetics of transnitrosation with nitrosoglutathione: Reactions with hemoglobin and other thiols", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS, SAN DIEGO, CA, US, vol. 254, no. 2, 15 December 1997 (1997-12-15), pages 215 - 220, XP002206812, ISSN: 0003-2697 *
SINGH RAVINDER JIT ET AL: "Mechanism of nitric oxide release from S-nitrosothiols", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 271, no. 31, 2 August 1996 (1996-08-02), pages 18596 - 18603, XP002206814, ISSN: 0021-9258 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009088801A1 (fr) * 2007-12-31 2009-07-16 Medical College Of Wisconsin Composés de piégeage de spin à base de nitrone bifonctionnel et trifonctionnel, et leurs utilisations
US8143420B2 (en) 2007-12-31 2012-03-27 Medical Collage of Wisconsin, Inc. Bifunctional and trifunctional nitrone spin trapping compounds and uses thereof
CN101893634A (zh) * 2009-05-20 2010-11-24 中国科学院生物物理研究所 特异检测蛋白质或多肽半胱氨酸巯基修饰的方法及其用途

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