Hcy structural analog aht that inhibits methionyl-tRNA synthetase to produce htl and its application

A technology with a similar structure and methionyl, applied in the biological field, can solve problems such as increased reactive oxygen species and increased Hcy levels

Inactive Publication Date: 2017-12-01
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But in vivo, because Met is the metabolic precursor of Hcy, it will lead to the increase of Hcy level, so Met can not be used as an inhibitor of HTL production in vivo, but this suggests that other structural analogs of Hcy may have the effect of inhibiting HTL production
[0008] The induction of oxidative stress and apoptosis is one of the main pathogenic mechanisms of hyperhomocysteinemia, but the specific mechanism is not very clear, but many reports show that HTL is the cause of hyperhomocysteinemia. The main factor leading to the increase of reactive oxygen species, therefore, we speculate that the Hcy structural analog AHT may reduce the generation of reactive oxygen species by reducing the production of HTL

Method used

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  • Hcy structural analog aht that inhibits methionyl-tRNA synthetase to produce htl and its application
  • Hcy structural analog aht that inhibits methionyl-tRNA synthetase to produce htl and its application
  • Hcy structural analog aht that inhibits methionyl-tRNA synthetase to produce htl and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Embodiment 1: the synthesis of AHT:

[0037]

[0038] Weigh 5 g DL-homocysteine ​​into a 250 mL round bottom flask, N 2 For replacement, add 160 mL of anhydrous methanol, and add SOCl dropwise under ice-cooling 2 4 mL, and stirred for 24 h at room temperature with the ice removed.

[0039] After stopping the reaction, the solvent was spin-dried. The crude product was dissolved in DCM, and 10.35 mL of Et 3 N, then join Boc 2 O 8.9 g, stirred at room temperature for 2.5 h.

[0040] After stopping the reaction, the solvent was spin-dried. The crude product plus CH 3 80 mL of OH, H 2 O 35 mL, add 9.2 mL of tributylphosphine dropwise, and stir at 50°C for 30 minutes. The reaction system with 200 mL ether, 100 mL H 2 O was diluted and the organic phase was separated. The aqueous phase was extracted twice with ether (2×100 mL), the organic phases were combined, washed with saturated brine, anhydrous Na 2 SO 4 After drying and spin-drying, a colorless oil (compo...

Embodiment 2

[0047] Example 2. Thin-layer chromatography experiments verify that AHT, a structural analogue of Hcy, can inhibit MARS from converting Hcy into HTL in vitro.

[0048] Reaction system configuration:

[0049] 50mM Hepes (pH8.0), 10mM MgCl 2 ,10mM 2-mercaptoethanol, 1.5mM ATP, 5mM homocysteine, 30μM methionyl tRNA synthetase, AHT, react at 25 for 100min

[0050] Thin Layer Chromatography Detection of HTL Formation

[0051] Spotting: Use a pencil to draw a straight line 2cm away from one end of the silica gel plate and make a spotting mark. Use a micropipette to draw 4 ul sample point to the marked sample addition point, the diameter of each point shall not be greater than 0.5cm, and dry it with cold air.

[0052] Development: prepare the developing agent according to n-butanol: glacial acetic acid: water = 4:1:1, mix evenly and pour it into the developing tank, place the spotted thin plate in the developing reagent color developing solution in the chromatography tank, and sea...

Embodiment 3

[0055] Example 3, the influence of AHT on the homocysteine ​​modification of total cell protein

[0056] HTL is the direct precursor of N-homocysteine ​​modification, and AHT, as an inhibitor of HTL production, can reduce the homocysteine ​​modification of total protein in cells, and can inactivate Hcy for total protein homocysteine ​​in cells The added effect of modification.

[0057] Selected cells: HEK293T and NIH3T3, culture conditions: DMEM containing 10% calf serum, 37°C, 5% CO 2 .

[0058] Drug treatment: when the cells were cultured to a confluence of about 80%, AHT was added to the culture medium, and at the same time, Hcy at a uniform concentration was added to both the AHT treatment group and the blank control group to simulate hyperhomocysteinemia. The treatment conditions are as follows surface:

[0059]

[0060] Sample collection and detection: After treating cells with Hcy for 4 hours, wash the cells with PBS 1-2 times, add 1×SDS loading buffer to collect ...

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Abstract

The invention belongs to the field of biotechnology, in particular to an Hcy structural analog Hcy structural analog that inhibits methionyl tRNA synthetase from producing HTL and an application thereof. The present invention uses homocysteine ​​(Hcy) structural analogs as inhibitors of methionyl tRNA synthetase to synthesize homocysteine ​​thiolactone (HTL), denoted as AHT. Through in vitro biochemical experiments, it was shown that AHT can inhibit the conversion of homocysteine ​​to homocysteine ​​thiolactone by methionyl tRNA synthetase; cell experiments showed that treating cells with this inhibitor significantly reduced the total intracellular total The level of homocysteine ​​modification of the protein and reduce the toxic effects of homocysteine ​​such as inducing oxidative stress and activating the Wnt / β-catenin signaling pathway; animal experiments show that AHT can reduce homocysteine ​​modification by reducing homocysteine. Therefore, the preventive effect on neural tube defects can be achieved, and there is no obvious toxicity to animals, and the inhibitor of the invention can be used to prepare a drug for treating diseases caused by hyperhomocysteinemia.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a Hcy structural analog and its application. The Hcy structural analog is used as an inhibitor for (species) methionyl tRNA synthetase to inhibit its conversion of homocysteine Converted into homocysteine ​​thiolactone (HTL), thereby inhibiting the modification of N-homocysteine ​​of protein; It also relates to the inhibitor and its acceptable derivatives in the preparation of prevention or treatment of high homocysteine Drug application in blood-related diseases. Background technique [0002] Hyperhomocysteinaemia (HHcy) refers to a condition in which the level of homocysteine ​​(Hcy) in the blood is abnormally increased. Hcy is an intermediate product of methionine (Met) metabolism. When folic acid or vitamin B6, vitamin B12 is deficient in the diet or genes related to homocysteine ​​metabolism are defective, the level of Hcy in the body increases. Hyperhomocysteinemia...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07C323/60A61K31/198A61P9/00A61P9/10A61P25/28A61P25/00A61P3/10A61P19/10
Inventor 赵世民徐薇王红艳梅馨予
Owner FUDAN UNIV
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