New conopeptide, medicinal composition and uses thereof

A technology of use and medicine, applied in the fields of biochemistry and molecular biology, can solve problems such as neuralgia that cannot be cured

Active Publication Date: 2016-10-05
HAINAN UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But these treatments can only have an analgesic eff

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  • New conopeptide, medicinal composition and uses thereof
  • New conopeptide, medicinal composition and uses thereof
  • New conopeptide, medicinal composition and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1: Sequence and artificial synthesis of new conotoxin peptide K41JM and its analogues

[0073] The amino acid sequence and nomenclature of K41JM and its analogues are shown in Table 1 below. The amino acid sequence in Table 1 can be entrusted to the company to carry out artificial synthesis, or the following methods can be used:

[0074] Resin peptides were artificially synthesized by Fmoc chemical method. Use reagent K (trifluoroacetic acid / water / ethanedithiol / phenol / thioanisole; 90:5:2.5:7.5:5, v / v / v / v / v) to cut the peptide from the resin, and use ice ether to precipitate and wash to recover The linear peptide crude product was purified with a preparative reverse HPLC C18 column (Vydac), and the elution linear gradient was 10-50% B60 within 40min. Solvent B was 60% ACN (acetonitrile), 40% H20, 0.92% TFA (trifluoroacetic acid); Solvent A is 1% TFA in water. The purified peptide was tested for purity with an analytical HPLC C18 column (Vydac), and its purit...

Embodiment 2

[0079] Example 2: Various subtypes of rat, mouse and human nAChRs in Xenopus oocytes expression in cells

[0080] Reference literature (Azam L, Yoshikami D, McIntosh JM.Amino acid residues that confer high selectivity of the alpha6 nicotinicacetylcholine receptor subunit to alpha-conotoxinMII[S4A,E11A,L15A].J Biol Chem.2008;283(17):11625-32 .) and the instructions of the in vitro transcription kit (mMessage mMachine in vitro transcription kit (Ambion, Austin, TX)) to prepare various rat neurotype nAChRs subtypes (α3β2, α6 / α3β2β3, α6 / α3β4, α9α10, α4β2, α4β4, α3β4, α2β2, α2β4, α7), human neuron nAChRs subtypes (α9α10, α6 / α3β2β3, α7), and cRNA of mouse and human muscle nAChRs (α1β1δε), the concentration of which was determined by UV 260nm OD value was calculated. Xenopus laveis oocytes (frog eggs) were collected by dissection, and cRNA was injected into the frog eggs, and the injected amount of each subunit was 5 ng cRNA. Inject 0.5-2.5 ng DNA per subunit of muscle nAChR. ...

Embodiment 3

[0081] Example 3: Experiment of Cono Peptide K41JM Blocking Various nAChRs Subtypes in Rats

[0082] One cRNA-injected frog egg was placed in a 30 μL Sylgard recording tank (diameter 4 mm × depth 2 mm), and the ND96 perfusate (96.0 mM NaCl, 2.0 mM KCl, 2.0 mM KCl, 1.8mM CaCl 2 ,1.0mM MgCl 2 , 5mM HEPES, pH 7.1-7.5) or ND96 (ND96A) containing 1mM atropine, the flow rate is 1ml / min. All conotoxin solutions also contained 0.1 mg / ml BSA to reduce non-specific adsorption of the toxin, and a switching valve (SmartValve, Cavro Scientific Instruments, Sunnyvale, CA) was used to freely switch between infusion of toxin or acetylcholine (ACh), And a series of three-way solenoid valves (solenoid valves, model 161TO31, Neptune Research, Northboro, MA) allow free switching between perfusion ND96 and ACh, etc. The Ach-gated current was recorded online when the two-electrode voltage-clamp amplifier (model OC-725B, Warner Instrument Corp., Hamden, CT) was set in the "slow" clamp and the c...

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Abstract

The invention belongs to the fields of biochemistry and molecular biology, relates to a new conopeptide, and a medicinal composition, a preparation method and uses thereof, also relates to a nucleic acid construct, an expression vector of the nucleic acid construct, a converted cell of the expression vector and a fusion protein of the conopeptide, and further relates to a method for blocking out an acetylcholine receptor, and pharmaceutical uses of the conopeptide. The new conopeptide K41JM and analogs thereof can specifically block out the acetylcholine receptor (nAChRs) (such as alpha9alpha10nAChR), have can treat neuralgia, cancer chemotherapy, breast cancer, lung cancer, wound healing, encephalomyelitis, epilepsy and ischaemia, and have good application prospects in the preparation of pain easing medicines, anticancer medicines and neuroscience tool medicines.

Description

technical field [0001] The invention belongs to the fields of biochemistry and molecular biology, and relates to a new conus peptide, its pharmaceutical composition, its preparation method and application. The invention also relates to analogs of said conopeptides. The present invention also relates to a method for blocking nicotinic acetylcholine receptors (nAChRs) and the pharmaceutical use of the conus peptide. Background technique [0002] Nicotinic acetylcholine receptors (nAChRs) are ion channel receptors ubiquitous in the animal kingdom, and contain such receptors from lower nematodes to higher mammals (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 the synapse and extrasynapse of the nerve-muscle (and / or) nerve-neuron junction, and activate the release of various neurotransmitters such as dopamine, norepinephri...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K1/20C07K1/06C12N15/12C12N15/63C07K19/00A61K38/08A61P25/02A61P25/16A61P35/00A61P17/02A61P29/00
CPCY02P20/55
Inventor 罗素兰长孙东亭朱晓鹏吴勇胡远艳李晓丹J·迈克尔·麦金托什戴维·J·克雷克
Owner HAINAN UNIVERSITY
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