Application of Cono Polypeptide bu8 and Its Derivatives

A technology of inhibition and products, applied in the field of biomedicine, can solve the problems of inability to judge the target, analgesic activity, and no analgesic activity

Active Publication Date: 2022-08-02
ACADEMY OF MILITARY MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Conotoxins containing the cysteine ​​framework structure "C-C-CC-C-C" also include conotoxins such as δ, μO, κ and γ families, which act on sodium and potassium ion channels and nicotinic acetylcholine receptors, etc. Except for the muO family toxins which have analgesic activity, the others have no analgesic activity, so the target and analgesic activity cannot be judged from the sequence and cysteine ​​skeleton structure

Method used

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  • Application of Cono Polypeptide bu8 and Its Derivatives
  • Application of Cono Polypeptide bu8 and Its Derivatives
  • Application of Cono Polypeptide bu8 and Its Derivatives

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Synthesis of Cono Polypeptide Bu8 and Its Derived Polypeptides

[0040] Conus polypeptide Bu8, derived from Conus bullatus, is also known as Bu8 polypeptide. Cono snail polypeptide MVIIA, derived from Cono snail (C. magus), is also called MVIIA polypeptide.

[0041] The following 13 polypeptides were prepared respectively (the C-terminus has been modified by acetylation):

[0042] Bu8 polypeptide (SEQ ID NO: 1 of the Sequence Listing): CKRKGSSCRRTSYDCCTGSCRNGKC;

[0043] Bu8[R3A] polypeptide (SEQ ID NO: 2 of the Sequence Listing): CK A KGSSCRRTSYDCCTGSCRNGKC;

[0044] Bu8[S6A] polypeptide (SEQ ID NO: 3 of the Sequence Listing): CKRKG A SCRRTSYDCCTGSCRNGKC;

[0045] Bu8[S7A] polypeptide (SEQ ID NO: 4 of the Sequence Listing): CKRKGS A CRRTSYDCCTGSCRNGKC;

[0046] Bu8[R9A] polypeptide (SEQ ID NO: 5 of the Sequence Listing): CKRKGSSC A RTSYDCCTGSCRNGKC;

[0047] Bu8[T11A] polypeptide (SEQ ID NO: 6 of the Sequence Listing): CKRKGSSCRR A SYDCCTGSCRNGKC; ...

Embodiment 2

[0069] Example 2. Structural determination of Cono polypeptide Bu8 and its derivatives

[0070] In this example, the solution three-dimensional structure of Cono polypeptide Bu8 was determined by solution nuclear magnetic resonance (NMR) method, and the secondary structure of Cono polypeptide Bu8 and its derivatives was determined by circular dichroism method.

[0071] 1. Solution NMR structure determination of Cono snail polypeptide Bu8

[0072] The solution NMR structure of Bu8 was determined by Professor Jiang Ling from the Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, and the determination method was the same as that in the literature (Yu, et al. Peptides. 2016, 81: 15-20). Bu8 dissolved in 90% H 2 O / 10%D 2 O (v / v; pH 3.5-4.0, pH adjusted with 50 mM sodium phosphate). NOESY, TOCSY and DQF-COSY were measured at 293K with a Bruker AVANCE III 850MHz NMR spectrometer. The distance constraint calculation includes 76 internal residue distances, 76 ...

Embodiment 3

[0077] Example 3. Target determination and activity evaluation of Cono snail polypeptide Bu8

[0078] In this example, the whole-cell patch-clamp experiment was used to determine the inhibitory activity of Cono polypeptide Bu8 on N-type calcium ion channels.

[0079] Experiments were performed using HEK293T cells. Cell culture conditions: 37°C, 5% CO 2 . Cell culture medium: Dulbecco's modified Eagle's medium (DMEM) medium containing 10% fetal bovine serum. Take a 24-well plate and plate HEK293T cells (about 2 × 10 per well). 5 cells), using Lipofectamine 2000 transfection reagent to co-transfect HEK293T cells with N-type calcium channel plasmid α1B, N-type calcium channel plasmid α2δ1, N-type calcium channel plasmid β and pEGFP-C1 plasmid (transfection per well). The total amount of plasmids was about 2 μg, and the mass ratio of the above four plasmids was 1:1:1:0.6), cultured for 24 hours, and then the cells were transferred to poly-L-lysine-coated glass coverslips And ...

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Abstract

The invention discloses the application of a Cono snail polypeptide Bu8 and its derivative polypeptides. The present invention provides the application of the specific polypeptide in the preparation of a product with analgesic function. The present invention also provides the application of the specific polypeptide in the preparation of a product having an inhibitory effect on N-type calcium ion channels. The specific polypeptide is the following (a) or (b): (a) the polypeptide shown in the sequence 1 of the sequence listing; (b) the polypeptide shown in the sequence 1 of the sequence listing is mutated by 1 to 3 amino acid residues The resulting derivative polypeptides. It is found in the present invention that the Cono snail polypeptide Bu8 and its derivative polypeptides selectively act on N-type calcium ion channels, have weak effects on other subtype calcium ion channels, have no obvious effect on voltage-gated sodium and potassium ion channels, and have significant inhibitory effects. pain activity, relatively low toxicity and adverse reactions.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular to the application of Cono polypeptide Bu8 and its derivative polypeptides, and more particularly to the application of Cono polypeptide Bu8 and its derivative polypeptides, which have the activity of inhibiting N-type calcium ion channels, in analgesia. Background technique [0002] Neuropathic pain is a pain syndrome caused by primary lesions or dysfunctions of the central or peripheral nervous system. Neuropathic pain can be caused by major diseases such as cancer, AIDS, and traumatic nerve injury, and presents typical symptoms such as paresthesia, hyperalgesia, and hyperalgesia. Clinically, the combination of anticonvulsants (such as leticital, oxcarbazepine), opioids (such as morphine, etc.) and antidepressants is often used to control severe neuropathic pain, but the adverse reactions caused by long-term use, Tolerance and addiction, often lead to poor treatment effect. N-type cal...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61P29/00A61P25/00C07K14/435
CPCA61K38/1767A61P29/00A61P25/00C07K14/43504
Inventor 戴秋云余硕陈金琴刘珠果
Owner ACADEMY OF MILITARY MEDICAL SCI
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