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Alpha9alpha10nAChR inhibitory active peptide and application thereof

A technology of active polypeptides and active monomers, which can be used in the fields of peptides, peptide sources, allergic diseases, etc., and can solve the problems of low yield, difficult synthesis, and difficult purification.

Pending Publication Date: 2022-06-03
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These disulfide bond isomers and multimers have different inhibitory activities on α9α10nAChR. It is difficult to synthesize during the production process, and it is possible to form the correct conformation through oxidative folding, which is difficult to purify and low yield.

Method used

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  • Alpha9alpha10nAChR inhibitory active peptide and application thereof
  • Alpha9alpha10nAChR inhibitory active peptide and application thereof
  • Alpha9alpha10nAChR inhibitory active peptide and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0101] Example 1: Synthetic method of polypeptide

[0102] The polypeptide in the present invention can be synthesized by Fmoc or Boc solid-phase synthesis method.

[0103] 1.1 Synthesis of linear peptides

[0104] Taking Fmoc solid-phase synthesis as an example, the synthesis steps are as follows:

[0105] 1. Weigh 0.2 mmol RAM resin, place it in a solid-phase reaction tube, and add equal proportions of N,N-dimethylformamide (DMF) and dichloromethane (DCM) to swell overnight;

[0106] 2. Add 20% piperidine and react in a shaking table at 37 degrees for 0.5h to remove the Fmoc protecting group of the amino group on the resin. After the reaction is completed, wash the resin three times with DMF and DCM respectively;

[0107] 4. Add 5eq of the C-terminal first amino acid, 4.5eq of HCTU and 8eq of DIPEA and place it on a shaker at 37 degrees to carry out the condensation reaction of the first amino acid. After the 1h reaction is completed, wash the resin three times with DMF an...

Embodiment 2

[0129] Example 2: Electrophysiological Activity Test Method

[0130] In order to verify the inhibitory activity of the active polypeptide on the human α9α10 acetylcholine receptor, the relative inhibitory intensity of the polypeptide on the acetylcholine-evoked peak current on the human α9α10 acetylcholine receptor was detected. Methods as below:

[0131] The cRNA of human α9α10 acetylcholine receptor was prepared by in vitro transcription kit, and its concentration was measured by OD value under UV260m. Xenopus oocytes were dissected and collected, and the cRNAs of the two subunits were injected into frog eggs on the first and second day, and the injection amount was 5ng, and cultured in ND-96. Oocytes expressing the α9α10 acetylcholine receptor were tested for electrophysiological activity 1-4 days after injection. The specific test method is as follows: 1 xenopus oocyte injected with cRNA was placed in a 30uL Sylgard recording tank with a diameter of 4mm and a depth of 2m...

Embodiment 3

[0132] Example 3: Construction of an animal model of in vivo analgesic activity

[0133] Animal models were used to evaluate the effect of active peptides on pain perception. The analgesic activity was tested using the rat sciatic nerve chronic compression injury (CCI) model.

[0134] The modeling process is as follows: after the rats were anesthetized by intraperitoneal injection of 2% pentobarbital sodium, the hair of the right leg was removed, the skin of the right lower limb was incised 1-2 cm under aseptic conditions, the muscles and fascia were bluntly separated, and the trunk of the sciatic nerve was exposed. , use 4-0 chrome catgut to tie four knots with a spacing of 1mm, and the tightness is required until the rat's toes twitch slightly, so as not to affect the blood supply of the epineurium, after the ligation is completed, use 40,000 units The wound was cleaned with sodium penicillin and finally sutured, and 40,000 units of sodium penicillin was intramuscularly inj...

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Abstract

The invention provides an alpha9alpha10 nicotinic acetylcholine receptor (nAChR) inhibitory active peptide and application thereof, a plurality of alpha-conotoxin monomer peptide mutants are designed according to the interaction mechanism of alpha-conotoxin and an alpha9alpha10 nicotinic acetylcholine receptor, and a monomer peptide mutant Gex-2 with high inhibitory activity and good analgesic effect is screened from the alpha-conotoxin monomer peptide mutants. And further performing mutation by means of alanine scanning, aspartic acid scanning, arginine scanning, arginine-citrulline substitution, D-type amino acid substitution and the like on the Gex-2 to obtain a series of derivatives of the Gex-2. The Gex-2 can improve the pain threshold / pain threshold value of a CCI rat, and has a good analgesic effect; compared with Gex-2, part of members in the series of Gex-2 derivatives have higher inhibitory activity on alpha9 alpha10nAChR (alpha 9 alpha 10 nAChR). The Gex-2 and the series of derivatives thereof can be used as alpha 9 alpha 10 nAChR inhibitors for analgesia, tumor resistance, nervous system disease treatment and the like.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, in particular to a polypeptide with analgesic activity and application thereof, in particular to a single-chain polypeptide with analgesic activity obtained by design, preparation and screening based on natural α-conotoxin and its mechanism of action Polypeptides and their derivatives. Background technique [0002] Nicotinic acetylcholine receptors (nAChRs) are ubiquitous ion channel receptors in the animal kingdom, ranging from lower nematodes to higher mammals containing such receptors (Nicke, A. (2004) Learning about structure and function of neuronal nicotinicacetylcholine receptors. Lessons from snails. European journal of biochemistry / FEBS 271, 2293). nAChRs receptors are located in and out of synapses at the nerve-muscle (and / or) nerve-nerve junction, and activate the release of various neurotransmitters such as dopamine, norepinephrine, serotonin, and gamma-aminobutyric acid. nAChRs me...

Claims

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Application Information

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IPC IPC(8): C07K14/435C07K1/06C07K1/04A61K38/17A61P29/00A61P35/00A61P25/00A61P25/30A61P25/16A61P25/08A61P25/28A61P39/02A61P25/02A61P3/10A61P31/22A61P17/02A61P31/18A61P9/14A61P13/12A61P1/16A61P11/00A61P43/00A61P31/04A61P31/14A61P31/20A61P3/02A61P3/06A61P31/08A61P19/02A61P37/08
CPCC07K14/43504A61P29/00A61P35/00A61P25/00A61P25/30A61P25/16A61P25/08A61P25/28A61P39/02A61P25/02A61P3/10A61P31/22A61P17/02A61P31/18A61P9/14A61P13/12A61P1/16A61P11/00A61P43/00A61P31/04A61P31/14A61P31/20A61P3/02A61P3/06A61P31/08A61P19/02A61P37/08A61K38/00A61K38/17C07K1/04C07K1/06C07K14/435C07K1/20C07K7/06A61K38/08C12N15/63
Inventor 于日磊李晓
Owner OCEAN UNIV OF CHINA
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