Spinosyn derivative as argininosuccinate synthetase activator and application of spinosyn derivative

A technology of argininosuccinic acid and argininosuccinic acid, which is applied in the field of medicine and achieves remarkable curative effect

Active Publication Date: 2020-09-08
CENT SOUTH UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there are no published reports of chemical small molecules that can regulate ASS1 activity.

Method used

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  • Spinosyn derivative as argininosuccinate synthetase activator and application of spinosyn derivative
  • Spinosyn derivative as argininosuccinate synthetase activator and application of spinosyn derivative
  • Spinosyn derivative as argininosuccinate synthetase activator and application of spinosyn derivative

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Target identification and confirmation experiments

[0067] The specific test steps are as follows:

[0068] 1) Synthesis of biotin-based spinosyn probe spinosyn-biotin complex Xn-03-17A.

[0069] (1) Synthesis of N-(3-bromohexyl) spinosyn: add 1 g (1.39mmoL) of spinosyn B, 20ml of acetonitrile and 385mg (1.39mmol) of potassium carbonate to a 100mL single-necked round bottom flask, and finally add 1,6-Dibromohexane 2.14 mL (13.9mmoL), stirred at 45°C for 48h. The resulting solid was removed by suction filtration, the solvent was removed, extracted with 50 mL of water and EA (3×60 mL), the combined organic phases were dried over anhydrous sodium sulfate. After removal of the solvent, 840 mg of white solid was obtained through silica gel column layer V (ethyl acetate): V (petroleum ether) = 4:1, yield 73%. m.p.95~98℃; HRMS calcd. for C 46 h 74 BrNO 10 880.4574, found 880.4602.

[0070] (2) Synthesis of spinosyn-hexyl-biotin (Xn-03-17A): Add DMF1.5 mL, compound 510...

Embodiment 2

[0075] Effects of Drugs on Protein ASS1 Enzyme Activity

[0076] The principle of ASS1 activity detection is: ASS1 catalyzes the reaction of citrulline and aspartic acid to generate argininosuccinic acid, which consumes ATP to generate pyrophosphate (PPi), and the generated PPi is produced under the catalysis of pyrophosphatase Phosphoric acid, phosphoric acid reacts with ammonium molybdate to generate phosphomolybdenum heteropolyacid, and under the reduction of vitamin C, it generates blue phosphomolybdenum blue, which has a characteristic absorption peak at OD660.

[0077] Enzyme activity calculation formula:

[0078] Enzyme activity is: [(drug+ASS1)OD 660 -NCOD 660 ] / (ASS1OD 660 -NCOD 660 )×100%.

[0079] (drug+ASS1)OD 660 : Solution absorbance (wavelength 660nm) when adding medicine and ASS1; ASS1OD 660 :

[0080] Solution absorbance when adding ASS1 Solution absorbance (wavelength 660nm); NCOD 660 : Blank reference solution absorbance (wavelength 660nm).

[0081...

Embodiment 3

[0098] Drugs against proteins ASS1 and ASS1 G362V Effect of enzyme activity

[0099] In order to detect SPA and its derivatives LM-2I on the protein ASS1 and ASS1 G362V Enzyme activity, we constructed the ASS1 pET28a plasmid through homologous recombination technology, and on this basis, used gene site-directed mutagenesis technology to construct ASS1 G362V pET28a plasmid, the constructed plasmid was transferred into BL21(DE3) for prokaryotic expression and protein purification of ASS1 and ASS1 G362V .

[0100] The specific steps are:

[0101] 3.1 ASS1 and ASS1 G362V amino acid sequence

[0102] The amino acid sequence of ASS1 is:

[0103]MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFMSSKGSVVLAYSGGLDTSCIL VWLKEQGYDVIAYLANIGQKEDFEEARKKALKLGAKKVFIEDVSREFVEEFIWPAIQSSALY EDRYLLGTSLARPCIARKQVEIAQREGAKYVSHGATGKGNDQVRFELSCYSLAPQIKVIAP WRMPEFYNRFKGRNDLMEYAKQHGIPIPVTPKNPWSMDENLMHISYEAGILENPKNQAPP GLYTKTQDPAKAPNTPDILEIEFKKGVPVKVTNVKDGTTHQTSLELFMYLNEVAGKHGVGR IDIVENRFIGMKSRGIYETPAGTILYHAHLDI...

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Abstract

The invention discloses spinosyn A and a derivative thereof as an activator of argininosuccinate synthetase 1 (ASS1) and an activator of mutant argininosuccinate synthetase ASS1G362V and application of the Spinosyn A and the derivative thereof. The spinosyn derivative has a structural general formula (I). The spinosyn and the derivative thereof are used as a medicine and an anti-tumor medicine fortreating I-type citrullinemia by activating the argininosuccinate synthetase 1 (ASS1) and the mutant argininosuccinate synthetase ASS1G362V in a targeted manner.

Description

technical field [0001] The invention relates to spinosyn A (Spinosyn A) and its derivatives which can regulate and activate argininosuccinate synthetase (Argininosuccinate synthetase 1, ASS1). The compound can be used as a treatment for diseases related to aminosuccinate synthase deficiency, such as anti-tumor and citrullinemia, and belongs to the field of medicine. Background technique [0002] Argininosuccinate synthase (ASS1; EC 6.3.4.5) was first discovered in the liver and later recognized as a ubiquitous enzyme in mammals. ASS1 gene is located on chromosome 9q34.11, the gene length is 56kb, the open reading frame length is 1239bp, has 16 exons, encodes 412 amino acids, and has a molecular weight of 46kDa. ASS1 catalyzes the generation of argininosuccinate from citrulline and aspartic acid under the condition of ATP function, and the compound is further decomposed into arginine and fumaric acid under the action of argininosuccinate lyase, in which arginine Amino acids...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/706A61K31/7048A61P3/00A61P35/00C12N9/00C12N15/70
CPCA61K31/706A61K31/7048A61P3/00A61P35/00C12N9/93C12N15/70C12Y603/04005
Inventor 罗志勇刘苏友邹自征胡息源罗眺陈筱丹孔繁蓉罗均利罗文松马大友
Owner CENT SOUTH UNIV
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