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Methods of diagnosing inflammatory diseases by determining pyroglutamate-modified mcp-1 and screening methods for inhibitors of glutaminyl cyclase

A technology of glutaminyl cyclase and MCP-1, which is applied in the fields of disease diagnosis, instrumentation, biological material analysis, etc.

Inactive Publication Date: 2013-02-27
VIVORYON THERAPEUTICS NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although plant QC appears to belong to a new enzyme family (Dahl, S.W. et al. 2000 Protein Expr Purif 20, 27-36), mammalian QC was found to share significant sequence homology with bacterial aminopeptidases (Bateman, R.C. et al. 2001 Biochemistry 40,11246-11250), leading to the conclusion that QCs from plants and animals have different evolutionary origins

Method used

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  • Methods of diagnosing inflammatory diseases by determining pyroglutamate-modified mcp-1 and screening methods for inhibitors of glutaminyl cyclase
  • Methods of diagnosing inflammatory diseases by determining pyroglutamate-modified mcp-1 and screening methods for inhibitors of glutaminyl cyclase
  • Methods of diagnosing inflammatory diseases by determining pyroglutamate-modified mcp-1 and screening methods for inhibitors of glutaminyl cyclase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0279] Embodiment 1: MALDI-TOF mass spectrometry

[0280] Matrix-assisted laser desorption / ionization mass spectrometry was performed using a Voyager De-Pro (Applied Biosystems, Darmstadt) equipped with a linear time-of-flight analyzer. The instrument is equipped with a 337nm nitrogen laser, a potential acceleration source and a 1.4m flying test tube. Detector operation is positive ion mode. Samples (5 μl) were mixed with an equal volume of matrix solution. For the matrix solution, sinapinic acid was used, which was prepared by dissolving 20 mg sinapinic acid (Sigma-Aldrich) in 1 ml acetonitrile / 0.1% TFA in water (1 / 1, v / v). Transfer a small volume (≈1 μl) of matrix-analyte mixture to the probe tip.

[0281] Test Glu for the long term 1 For cyclization, the MCP-1 peptide was incubated at 30° C. in 100 μl of 0.1 M sodium acetate buffer, pH 5.2 or 0.1 M Bis-Tris buffer, pH 6.5. Peptides were administered at a concentration of 0.15mM to 0.5mM and 0.2U QC was added. At vario...

Embodiment 2

[0282] Example 2: Proteolysis of human MCP-1(1-76) by dipeptidyl peptidase 4 (DP4), aminopeptidase P and proteases present in human serum

[0283] N-terminus of MCP-1 by recombinant human DP4 in the presence and absence of QC-specific inhibitors terminal degradation

[0284] Recombinant human MCP-1 starting with N-terminal glutamine (1-76) (SEQ ID NO: 1) (Peprotech) was dissolved in 25 mM Tris / HCl pH 7.6 at a concentration of 10 μg / ml. The MCP-1 solution was pre-incubated with recombinant human QC (0.0006 mg / ml) at 30°C for 3 hours, followed by incubation with recombinant human DP4 (0.0012 mg / ml) at 30°C, or with DP4 without prior addition of QC. In addition, Gln 1 -Incubation of MCP-1 with recombinant human QC in the presence of 1-(3-(1H-imidazol-1-yl)propyl)-3-(3,4-dimethoxyphenyl)thiourea hydrochloride conduct. The resulting DP4 cleavage products were analyzed by Maldi-TOF mass spectrometry after 0 min, 15 min, 30 min, 1 h, 2 h and 4 h.

[0285] N-terminal degrad...

Embodiment 3

[0290] Example 3: Degradation of Human MCP-2, MCP-3 and MCP-4

[0291] N-terminal degradation of human MCP-2 by DP4

[0292] Human recombinant MCP-2 (Peprotech) carrying an N-terminal glutaminyl rather than pyroglutamyl residue was dissolved in 25 mM Tris / HCl, pH 7.6 at a concentration of 10 μg / ml. MCP-2 was pre-incubated with recombinant human QC (0.0006mg / ml) at 30°C for 3 hours, then incubated with recombinant human DP4 (0.0012mg / ml) at 30°C or not pre-incubated with QC but with recombinant human DP4 (0.0012mg / ml) insulation. The resulting DP4 cleavage products were analyzed by Maldi-TOF mass spectrometry after 0 min, 15 min, 30 min, 1 h, 2 h, 4 h and 24 h.

[0293] N-terminal degradation of human MCP-3 by DP4

[0294] Human recombinant MCP-3 (SEQ ID NO: 12) (Peprotech) carrying an N-terminal glutaminyl rather than pyroglutamyl residue was dissolved in 25 mM Tris / HCl, pH 7.6 at a concentration of 10 μg / ml. MCP-3 was pre-incubated with recombinant human QC (0.0006mg...

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Abstract

The invention relates to a method to monitor treatment of an inflammatory disease or an inflammatory associated disease with the use of the ratio of N-terminal pyroglutamate modified MCP-1 (MCP-1 N1pE) : total concentration of MCP-1 within a biological sample as a biomarker and further concerns a novel method to determine the proportion of N-terminal pyroglutamate modified MCP-1 in relation to the total concentration of MCP-1 in biological samples. The invention also provides a diagnostic kit and a method for screening a glutaminyl cyclase (QC) inhibitor or measuring the effectiveness of a glutaminyl cyclase (QC) inhibitor.

Description

field of invention [0001] The present invention relates to a method for monitoring the treatment of inflammatory diseases or inflammation-related diseases using the ratio of N-terminal pyroglutamate-modified MCP-1 (MCP-1N1pE): MCP-1 total concentration in a biological sample as a biomarker, It further relates to a novel method for determining the ratio of N-terminal pyroglutamate-modified MCP-1 relative to the total concentration of MCP-1 in a biological sample. The present invention also provides diagnostic kits and methods for screening glutaminyl cyclase (QC) inhibitors or measuring the efficacy of glutaminyl cyclase (QC) inhibitors. Background of the invention [0002] Chemotactic cytokines (chemokines) are proteins that attract and activate white blood cells and are thought to play an important role in inflammation. Chemokines are divided into 4 families (C-, CC-, CXC- and CX3C-chemokines) by the appearance of the N-terminal cysteine ​​residue. CC-chemokines (aka β-ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/68
CPCG01N2800/2814G01N2800/102G01N33/6893G01N33/574
Inventor H·辛尼斯M·克莱因施密特K·甘斯J-U·拉费尔德H-U·德穆特N·陶特
Owner VIVORYON THERAPEUTICS NV
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