α-conotoxin peptide, pharmaceutical composition and use thereof

Patent metadata

Field	Value
Publication number	US-9469674-B2
Application number	US-201314419584-A
Country	US
Kind code	B2
Filing date	Jun 18, 2013
Priority date	Aug 7, 2012
Publication date	Oct 18, 2016
Grant date	Oct 18, 2016

Abstract

The present invention provides a novel α-conotoxin peptide, pharmaceutical composition and use thereof. The present invention further provides a propeptide of the conotoxin peptide, a nucleic acid construct, expression vector and transformed cell of the conotoxin peptide as well as a fused protein of the conotoxin peptide. The present invention discloses a method for blocking acetylcholine receptors as well as a use of the conotoxin peptide in the manufacture of a medicament. The α-conotoxin peptide of the present invention can specifically block acetylcholine receptor (nAChRs, such as α3β2 nAChRs, α6/α3β2β3 nAChR or α3β4 nAChR or α6/α3β4 nAChR), has activity for treatment of neuralgia, addiction, Parkinson's disease, dementia, schizophrenia, cancers, and can be used in the manufacture of a medicament for analgesia and smoking cessation and drug-withdrawal, a medicament for treatment of mental diseases and cancers, as well as a tool drug for neurosciences.

First claim

What is claimed is:

1. A polypeptide, which comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12, wherein the carboxyl terminal of the polypeptide is amidated.

2. The polypeptide according to claim 1 , wherein at the N-terminal of the polypeptide, the 1st cysteine and the 3rd cysteine of said amino acid sequence form a disulfide bond, and the 2nd cysteine and the 4th cysteine of said amino acid sequence form a disulfide bond; or at the N-terminal of the polypeptide, the 1st cysteine and the 4th cysteine of said amino acid sequence form a disulfide bond, and the 2nd cysteine and the 3rd cysteine of said amino acid sequence form a disulfide bond; or at the N-terminal of the polypeptide, the 1st cysteine and the 2nd cysteine of said amino acid sequence form a disulfide bond, and the 3rd cysteine and the 4th cysteine of said amino acid sequence form a disulfide bond.

3. The polypeptide according to claim 1 , wherein the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 11 and SEQ ID NO: 12.

4. The polypeptide according to claim 3 , wherein at the N-terminal of the polypeptide, the 1st cysteine and the 3rd cysteine of said amino acid sequence form a disulfide bond, and the 2nd cysteine and the 4th cysteine of said amino acid sequence form a disulfide bond; or at the N-terminal of the polypeptide, the 1st cysteine and the 4th cysteine of said amino acid sequence form a disulfide bond, and the 2nd cysteine and the 3rd cysteine of said amino acid sequence form a disulfide bond; or at the N-terminal of the polypeptide, the 1st cysteine and the 2nd cysteine of said amino acid sequence form a disulfide bond, and the 3rd cysteine and the 4th cysteine of said amino acid sequence form a disulfide bond.

5. A pharmaceutical composition, which comprises the polypeptide according to claim 1 and a pharmaceutically acceptable carrier or excipient.

6. A method for blocking an acetylcholine receptor or regulating acetylcholine level in cells, comprising the step of applying an effective amount of the polypeptide according to claim 1 to the cells, wherein said acetylcholine receptor is α6/α3β2β3 acetylcholine receptor.

7. The method of claim 6 , wherein the cells are cells in vivo.

8. A method for preparing the polypeptide according to claim 1 , comprising the following steps:
1) synthesizing a linear polypeptide by ABI Prism 433a polypeptide synthesizer or by a manual method, in which side-chain protecting groups of Fmoc amino acids are: Pmc (Arg), Trt or Acm (Cys), But (Thr, Ser, Tyr), OBut (Asp), and Boc (Lys);
2) cutting the linear polypeptide of step 1) from resin;
3) using glacial diethyl ether to precipitate and wash the linear polypeptide obtained in step 2), and recovering a crude product of linear polypeptide;
4) using a preparative reversed phase HPLC C18 column to purify the crude product of linear polypeptide obtained in step 3); and
5) subjecting the product obtained in step 4) to two-step or one-step oxidative folding.

Assignees

• Univ Hainan

Inventors

• Luo Sulan
• Zhangsun Dongting
• Wu Yong
• Zhu Xiaopeng
• Hu Yuanyan
• Bing Hui
• Mcintosh J Michael

Classifications

• A61P35/00

  Antineoplastic agents · CPC title

• A61P25/18

  Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia · CPC title

• C07K14/43504 Primary

  from invertebrates · CPC title

• C07K1/061

  using protecting groups · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title