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Medical use of inhibitors of glutaminyl and glutamate cyclases

A technique for inhibitors of glutaminyl cyclase and aminopeptidase, applied in the field of effector screening

Inactive Publication Date: 2010-09-22
VIVORYON THERAPEUTICS NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Not yet determined in vivo [pGlu 3 ] Cyclization mechanism of the 3-position N-terminal glutamic acid of the natural precursor of Aβ (Shirotani, K., Tsubuki, S., Lee, H.J., Maruyama, K., and Saido, T.C. (2002) Neurosci Lett 327, 25 -28)

Method used

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  • Medical use of inhibitors of glutaminyl and glutamate cyclases
  • Medical use of inhibitors of glutaminyl and glutamate cyclases
  • Medical use of inhibitors of glutaminyl and glutamate cyclases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0279] Embodiment 1: the preparation of people and papaya QC

[0280] Host Strains and Media

[0281] Pichia pastoris strain X33 (AOX1, AOX2) for human QC expression was grown, transformed and analyzed according to the manufacturer's instructions (Invitrogen). The media required for P. pastoris, buffered glycerol (BMGY) complex or methanol (BMMY) complex medium and fermentation basal salt medium, were prepared according to the manufacturer's recommendations.

[0282] Molecular cloning of a plasmid vector encoding human QC

[0283] All cloning steps are performed using standard molecular biology techniques. The vector pPICZαB (Invitrogen) was used for expression in yeast. The pQE-31 vector (Qiagen) was used to express human QC in E. coli. The cDNA for the mature QC starting at codon 38 was fused in-frame to a plasmid-encoded 6×histidine tag. After amplification and subcloning using primers pQCyc-1 and pQCyc-2 (Table 1), the fragment was inserted into the expression vec...

Embodiment 2

[0296] Example 2: Determination of glutaminyl cyclase activity

[0297] Fluorescence analysis

[0298] All assays were performed at 30°C using a BioAssay Reader HTS-7000Plus microplate reader (Perkin Elmer). QC activity was assessed fluorimetrically using H-Gln-βNA. The sample was prepared with 0.25 U pyroglutamyl aminopeptidase (Unizyme, Denmark) and appropriately diluted QC aliquots in a final volume of 250 μl. The excitation / emission wavelengths are 320 / 410nm. The assay reaction was initiated by the addition of glutaminyl cyclase. QC activity was determined from a β-naphthylamine standard curve under analytical conditions. One unit is defined as the amount of QC that catalyzes the generation of 1 μmol pGlu-βNA from H-Gln-βNA per minute under the stated conditions.

[0299] In the second fluorescence assay, the activity of QC was determined using H-Gln-AMC as substrate. Reactions were performed at 30°C using a NOVOStar microplate reader (BMG labtechnologies). The ...

Embodiment 3

[0305] Embodiment 3: MALDI-TOF mass spectrometry analysis

[0306] Matrix-assisted laser desorption / ionization mass spectrometry was performed using a Hewlett-Packard G2025LD-TOF System with a linear time-of-flight analyzer. The device is equipped with a 337nm nitrogen laser, a voltage acceleration source (5kV) and a 1.0m flight tube. The detector was in positive ion mode, and a LeCroy 9350M digital storage oscilloscope connected to a personal computer was used to record and filter the signal. Samples (5 μl) were mixed with an equal volume of matrix solution. For the matrix solution, we used an aqueous solution (1 / 1, v / DHAP / DAHC prepared in v). A small volume (=1 μl) of matrix-analyte-mixture was transferred onto the probe tip and evaporated immediately in a vacuum chamber (Hewlett-Packard G2024A sample preparation accessory) to ensure fast and uniform sample crystallization.

[0307] For long-term determination of Glu 1 For cyclization, Aβ-derived peptides were incubate...

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Abstract

The present invention provides novel physiological substrates of mammalian glutaminyl cyclase (QC, EC 2.3.2.5), new effectors of QC, methods for screeing for such effectors, and the use of such effectors and pharmaceutical compositions comprising such effectors for the treatment of conditions that can be treated by modulation of QC-activity. Preferred compositions additionally comprise inhibitors of DP IV or DP IV-like enzymes for the treatment or alleviation of conditions that can be treated by modulation of QC- and DP IV-activity.

Description

[0001] This application is a divisional application of the Chinese patent application No. 200480012386.2 filed on May 5, 2004, entitled "Application of glutaminyl and glutamate cyclase effectors". technical field [0002] The present invention relates to glutaminyl cyclase (glutaminyl cyclase) (QC, EC 2.3.2.5), which catalyzes the conversion of N-terminal glutamine residues into pyroglutamic acid (5-oxo-proline, pGlu*) with the simultaneous release of ammonia and the conversion of the N-terminal glutamic acid residue to pyroglutamic acid with the release of water molecules. [0003] The present invention confirms that the QC of mammals is a metalloenzyme, provides the application of novel physiological substrates and QC effectors of mammalian QCs, and is used for the treatment of diseases that can be treated by regulating QC activity. A pharmaceutical composition comprising QC effectors . Furthermore, metal interaction has been found to be an effective approach to develop QC ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K45/06A61P1/04A61P35/00A61P17/06A61P19/02A61P19/04A61P29/00A61P9/10A61K31/00A61K31/4178A61K31/4184A61K31/4188A61K31/4192A61K31/4745C07D231/12C07D233/54C07D403/06C07D405/06C07D409/06C07D471/04C07F9/572C07F9/59C07F9/6506C07F9/6561
CPCC07D471/04C07D233/54C07F9/6561A61K31/4192A61K31/4188C07D409/06A61K31/4178C07F9/597C07D405/06C07F9/5726A61K31/00C07D231/12C07F9/65066C07D403/06A61K31/4184C07F9/572C07F9/59C07F9/6506A61P1/04A61P17/06A61P19/02A61P19/04A61P25/00A61P25/28A61P29/00A61P35/00A61P9/10A61K31/4745
Inventor 汉斯-乌尔里希·德穆特托尔斯滕·霍夫曼安德烈·J·尼斯特罗杰斯特凡·席林乌尔里希·海泽
Owner VIVORYON THERAPEUTICS NV
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