WO2005081622A1

WO2005081622A1 - Methods and compositions for the detection of nitric oxide

Info

Publication number: WO2005081622A1
Application number: PCT/IB2005/000301
Authority: WO
Inventors: B. Mason Hughes; Christine Kornmeier; Thomas Misko; Barnett Pitzele
Original assignee: Pharmacia & Upjohn Company Llc
Priority date: 2004-02-17
Filing date: 2005-02-07
Publication date: 2005-09-09

Abstract

A method of detecting S-nitrosylated proteins or peptides in a biological sample, said method comprising: photolytically cleaving S-nitrosothiols in said S-nitrosylated proteins or peptides to form thiyl radical groups; contacting said biological sample with a thiyl radical-reacting compound, said thiyl radical-reacting compound modified with at least one marker; and detecting said marker with an appropriate detecting device.

Description

Methods and Compositions for the Detection of Nitric Oxide

BACKGROUND OF THE INVENTION The inorganic molecule nitric oxide (NO) exerts pleiotropic effects including smooth muscle relaxation, apoptosis, neurotransmitter release, neurotoxicity, and differentiation. In mediating vasorelaxation, NO stimulates cGMP formation by binding to the Fe heme center at the active site of soluble guanylyl cyclase which leads to a conformational alteration that augments enzyme activity. Another important mechanism by which these effects of NO are mediated appears to be through a chemical modification of cysteine residues, termed S-nitrosation or S-nitrosylation . 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. The reversible regulation of protein function by S-nitrosylation has led to suggestions that nitrosothiols function as posttranslational modifications analogous to phosphorylation or acetylation. The bulk of the evidence for protein regulation by S-nitrosylation has relied on in vitro experiments with NO donors, which in some cases also release other reactive oxygen species, or release NO molecules that differ in electronic structure from NO formed by nitric oxide synthase (NOS). Additionally, the cytoplasm contains high concentrations of glutathione and metals, which can bind NO, making it unclear whether S-nitrosylation can be elicited by endogenously produced NO. Nonetheless, airway S-nitrosothiols have been measured in patients suffering from severe asthma, and S- nitrosoproteins have been detected in serum and synovial fluid taken from rheumatoid arthritis patients and multiple sclerosis patients. 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 (5,5 '-dimethyl- 1-ρyrroline N-oxide), PBN (phenyl-N-t-butylnitrone), and BMPO (5-tert-butoxycarbonyl 5-methyl-l-pyrroline N-oxide) are spin traps normally used to detect oxygen centered radicals and thiyl radicals by Electron Paramagnetic Resonance (EPR). Upon photolytic decomposition of S-nitrosothiols the resultant thiyl radical will form a specific and stable adduct with DMPO (PBN, BMPO). Similarly 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. Such adducts are typically observed by EPR but may also be identified and quantitated by mass spectrometry. S-nitroso-proteins and peptide adducts can be identified in mass spectrometry by their mass shift, and exact neutral loss.

SUMMARY OF THE INVENTION

Light exposure will drive the decomposition of S-nitrosothiols into thiyl radicals and nitric oxide according to the equation: hv RSNO → RS^* + NO^*

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, ^l4C 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. -J-

Similarly, fluorescent tagging or radiolabelling (such as, for example ^l4C 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.

DETAILED DESCRIPTION OF THE INVENTION Definitions The term "DMPO," as used herein, 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

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- Δ 1-pyrroline 1 - oxide; 5, 5-Dimethyl-Δ 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. The term, "BMPO," as used herein, 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. The term "PBN," as used herein, means 2-Propanamine, 2-methyl-N- (phenylmethylene)-, N-oxide (9CI) , CAS Registry number 3376-24-7. PBN has the following structure, Ph represents phenyl and Bu-t represent tert-butoxycarbonyl: h — CH = N— Bu-t

PBN is also sometimes known as Nitrone, N-tert-butyl-α-phenyl- (6CI, 7CI, 8CI); α- Phenyl-N-tert-butylnitrone; α-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-α-phenylnitrone; N-tert- Butyl-2-phenylnitrone; N-tert-Butyl-C-phenylnitrone; PBN (amine oxide); and tert- Butyl(benzylidene)amine N-oxide. The term "POBN," as used herein, means 2-Propanamine, 2-methyl-N-[(l- oxido-4-pyridinyl)methylene]-, N-oxide (9CI) , CAS registry number 66893-81-0. POBN has the structure:

POBN is also sometimes called 2-Propanamine, 2-methyl-N-(4- pyridinylmethylene)-, N,N'-dioxide; α-(4-Pyridyl-l-oxide)-N-tert-butylnitrone; 4- POBN; C-(4-Pyridinyl-N-oxide)-N-tert-butylnitrone; and N-tert-Butyl-α-(4-pyridyl-l- oxide) nitrone The term "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-(3aα,4β,6aα)]-; (+)-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. The term "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. The term "hv," as used herein, 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. In another embodiment of the present invention, ultraviolet radiation may be used, wherein the wavelength (λ) is between 380 and 3000 nm (nanometers), and the frequency (v) is between 7.9 x 10 and lx 10¹⁶ 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. Where a fluorescent marker is employed, a UV-visual spectrometer may be utilized. Immunohistochemistry techniques could be employed to detect the thiyl radicals in tissue. These are merely illustrative. Example: DMPO- labeling of Nitrosothiols

Protein — S — NO _► NO ^■ Protein — S • Biotinylated-DMPO Biotin sM,. - ^■ Protein

In a broad sense, 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. In an exemplary embodiment of the invention, 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. In a preferred embodiment, the spacer is a Ci - C₈ 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. From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims

CLAIMSWhat is claimed is:

1. A method of selectively and specifically labeling S-nitrosothiols comprising performing the following steps: providing a thiyl radical-reacting compound, said thiyl radical-reacting compound modified with at least one marker; photolytically cleaving said S-nitrosothiols to form thiyl radical groups; and contacting said modified thiyl radical-reacting compound with said thiyl radical groups, thereby forming a stable adduct.

2. The method of claim 1 wherein said thiyl radical-reacting compound is selected from the group consisting of DMPO, EMPO, PBN and BMPO.

3. The method of claim 1 wherein said at least one marker is selected from the group consisting of a fluorescent marker, a radioactive isotope, hexa-histidine and biotin.

4. The method of claim 3 wherein said at least one marker is biotin.

5. The method of claim 3 wherein said thiyl radical-reacting compound is DMPO.

6. The method of claim 4 wherein said thiyl radical-reacting compound is DMPO.

7. A method of detecting S-nitrosylated proteins or peptides in a biological sample, said method comprising: photolytically cleaving S-nitrosothiols in said S-nitrosylated proteins or peptides to form thiyl radical groups; contacting said biological sample with a thiyl radical-reacting compound, said thiyl radical-reacting compound modified with at least one marker; and detecting said marker with an appropriate detecting device.