Small peptide and application thereof in inhibiting opiate addiction and tolerance
阅读说明：本技术 一种小肽及其在抑制阿片成瘾性和耐受性上的应用 (Small peptide and application thereof in inhibiting opiate addiction and tolerance ) 是由 周培岚 苏瑞斌 张艺馨 卢凤凤 于 2020-05-26 设计创作，主要内容包括：本发明涉及涉及一种小肽及其在抑制阿片成瘾性和耐受性上的应用,所述小肽氨基酸序列为:N’-Leu-Lys-Gln-Gln-Val-Lys-Ile-Phe-Glu-Glu-Asp-Phe-Gln-Arg-Glu-Arg-Ser-Asp-Arg-Glu-Arg-C’。本发明还提供一种具有抑制阿片成瘾和耐受性的药品,其含有的抑制阿片成瘾和耐受性的活性成分为上述小肽。本发明明确给出具有抑制阿片成瘾和耐受性的活性成分的小肽的氨基酸序列,其中的氨基酸可以由功能类似的同性氨基酸进行替换。本发明提供的小肽及修饰过的短肽,能显著提高阿片的镇痛效果,延长镇痛时间,抑制阿片耐受作用,阿片镇痛耐受被抑制则成瘾性相应的也被抑制。本发明还提出可通过固相合成法人工合成该小肽,使之有望发展成为抗阿片成瘾肽并能批量生产。(The invention relates to a small peptide and application thereof in inhibiting opiate addiction and tolerance, wherein the amino acid sequence of the small peptide is N '-Leu-Lys-Gln-Gln-Val-Lys-Ile-Phe-Glu-Glu-Asp-Phe-Gln-Arg-Glu-Arg-Ser-Asp-Arg-Glu-Arg-C'. The invention also provides a medicine for inhibiting opiate addiction and tolerance, which contains the active ingredient for inhibiting opiate addiction and tolerance as the small peptide. The invention specifically provides an amino acid sequence of a small peptide with an active ingredient for inhibiting opiate addiction and tolerance, wherein the amino acid can be replaced by functionally similar homologous amino acid. The small peptide and the modified short peptide provided by the invention can obviously improve the analgesic effect of opium, prolong the analgesic time and inhibit the tolerance of opium, and the addiction is correspondingly inhibited if the analgesic tolerance of opium is inhibited. The invention also provides a method for artificially synthesizing the small peptide by a solid phase synthesis method, so that the small peptide is expected to be developed into the anti-opioid addiction peptide and can be produced in batches.)
1. A small peptide characterized by the amino acid sequence of said small peptide
Is N '-Leu-Lys-Gln-Gln-Val-Lys-Ile-Phe-Glu-Glu-Asp-Phe-Gln-Arg-Glu-Arg-Ser-Asp-Arg-Glu-Arg-C'.
2. The small peptide of claim 1, wherein one or more amino acids in the amino acid sequence are replaced with functionally similar homologous amino acids as follows: from the N end, Leu at the 1 st position is replaced by one of Ala, Met, Pro, Val or Ile, Val at the 5 th position is replaced by one of Ala, Met, Pro, Leu or Ile, Phe at the 8 th position is replaced by Trp or Tyr, Glu at the 9 th position is replaced by Asp, Asp at the 11 th position is replaced by Glu, Glu at the 15 th position is replaced by Asp, Arg at the 16 th position is replaced by Lys or His, and Asp Glu at the 18 th position is replaced by Glu.
3. The small peptide of claim 1 or 2, wherein the small peptide is linked to a membrane-penetrating peptide to obtain a modified short peptide, and the membrane-penetrating peptide is linked to the N-terminus or C-terminus of the small peptide through a covalent bond.
4. The modified short peptide according to claim 3, wherein the cell-penetrating peptide is linked to the N-terminus of the small peptide through a covalent bond.
5. The modified short peptide according to claim 3 or 4, wherein the cell-penetrating peptide is TAT and has the amino acid sequence:
N’-Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-GlnC’。
6. a drug having an inhibitory activity against opiate addiction and opiate tolerance, characterized in that it contains the small peptide according to claim 1 or 2 as an active ingredient against opiate addiction and opiate tolerance.
7. A pharmaceutical product having an opioid addiction and tolerance inhibiting activity, which comprises the modified short peptide of any one of claims 3 to 5 as an active ingredient.
8. The drug product for inhibiting opiate addiction and opiate tolerance according to claim 6 or 7, wherein the drug product is an injection and is administered intravenously or intracerebroventricularly.
9. Use of one of claims 1 to 8 for the inhibition of opiate addiction and tolerance.
10. The small peptide according to claim 1 or 2, characterized in that the synthetic method is a solid phase synthesis method, comprising the steps of firstly binding the first amino acid with an amino acid protecting agent and resin, then removing the first amino acid protecting group, then binding the second amino acid with a condensation reagent, and carrying out the same operation until the last amino acid is bound, and purifying by HPLC.
Technical Field
The invention relates to the field of medicines, in particular to a small peptide and application thereof in inhibiting opiate addiction and tolerance.
Background
Opioid drugs have strong analgesic effect and are abused in a large number in some countries, but opioid drugs are easy to generate tolerance and dependence, so that the opioid drug crisis which is the focus of the world now is caused, the substance has strong addiction, serious withdrawal symptoms and high recurrence rate, most of students at home and abroad refer to opioid addiction as drug-dependent encephalopathy at present, and the radical treatment is very difficult.
In order to clarify the mechanism of opiate addiction and solve opiate dependence tolerance, a large amount of research is carried out on the non-opiate receptor action system and the opiate receptor action system per se by scholars at home and abroad, and important progress is made. For example, it is now known that the Mu Opioid Receptor (MOR) is a key receptor for mediating opioid analgesia and addiction, and foreign researchers have screened a series of MOR interacting proteins: silk protein a (filamin a), Periplakin, PKCI, RGS4, PLD2 and the like, which interact with opioid receptors to affect the activation effect of agonists on opioid receptors, further suggesting that opioid receptors are not completely restricted to coupling with recognized G proteins after activation, and that there may be more or more specific proteins involved in the regulation of opioid receptors, of which the ABIN1 protein is likely to be one. ABIN1, also known as TNIP1 (TNFa-induced protein 3 (TNAIP 3) -interacting
But further studies, such as what groups of ABIN1 interact with mu opioid receptors? By what mechanism? How to screen out effective active groups and successfully apply the active groups to medicines has not been reported.
Disclosure of Invention
The invention aims to provide a small peptide and application thereof in inhibiting opiate addiction and tolerance, and solves the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a small peptide, wherein the amino acid sequence of the small peptide is N '-Leu-Lys-Gln-Gln-Val-Lys-Ile-Phe-Glu-Glu-Asp-Phe-Gln-Arg-Glu-Arg-Ser-Asp-Arg-Glu-Arg-C'.
Further, one or more amino acids in the amino acid sequence may be replaced by functionally similar homologous amino acids, as follows: from the N end, Leu at the 1 st position is replaced by one of Ala, Met, Pro, Val or Ile, Val at the 5 th position is replaced by one of Ala, Met, Pro, Leu or Ile, Phe at the 8 th position is replaced by Trp or Tyr, Glu at the 9 th position is replaced by Asp, Asp at the 11 th position is replaced by Glu, Glu at the 15 th position is replaced by Asp, Arg at the 16 th position is replaced by Lys or His, and Asp Glu at the 18 th position is replaced by Glu.
Further, the invention provides a modified short peptide, which is obtained by connecting the small peptide with a cell-penetrating peptide, wherein the cell-penetrating peptide is connected with the N end or the C end of the small peptide through a covalent bond.
Further, the cell-penetrating peptide is connected with the N end of the small peptide through a covalent bond.
Further, the cell-penetrating peptide is TAT, and the amino acid sequence of the cell-penetrating peptide is as follows:
Tyr-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln。
the invention also provides a medicine for inhibiting opiate addiction and tolerance, which contains the active ingredient for inhibiting opiate addiction and tolerance as the small peptide.
Further, the invention provides a medicine for inhibiting opiate addiction and tolerance, which contains the active ingredient for inhibiting opiate addiction and tolerance as the modified short peptide.
The medicine provided by the invention can inhibit opiate addiction and tolerance, is an injection and is administered intravenously or intracerebroventricularly.
The small peptide, the modified short peptide and the medicine with the functions of inhibiting the addiction and the tolerance of the opium are all applied to inhibiting the addiction and the tolerance of the opium.
The invention also provides the artificial synthesis method of the small peptide, which adopts a solid-phase synthesis method and comprises the steps of firstly combining the first amino acid with the amino acid protective agent and resin, then removing the first amino acid protective group, then combining the condensation reagent with the second amino acid, carrying out the same operation until the last amino acid is combined, and purifying by HPLC.
The invention has the beneficial effects that:
1. the amino acid sequence of a small peptide with an active ingredient inhibiting opiate addiction and tolerance is clearly given: the prior art only investigated that ABIN1 is an interacting protein of the Mu Opioid Receptor (MOR) with two active domains AHD and UBAN, but the current results of the study alone did not meet the requirements for drug production. According to the invention, the AHD region is divided into 4 spliceosomes AHD1/AHD2/AHD3/AHD4 for research, and the 21 amino acid residue sequence of AHD2 is an active region with addiction resistance and tolerance resistance, and the small peptide can be tightly combined with hMOR1 protein through interaction such as hydrogen bond, salt bridge, pi-cation and hydrophobic interaction. Therefore, the technical scheme of the invention clearly provides that the active ingredient for inhibiting the addiction and tolerance of the opium is a small peptide, and the amino acid sequence of the small peptide is N '-Leu-Lys-Gln-Gln-Val-Lys-Ile-Phe-Glu-Glu-Asp-Phe-Gln-Arg-Glu-Arg-Ser-Asp-Arg-Glu-Arg-C', wherein the amino acid can be replaced by the homologous amino acid with similar function. The invention also provides a method for artificially synthesizing the small peptide by a solid phase synthesis method, so that the small peptide is expected to be developed into the anti-opioid addiction peptide and can be produced in batches.
2. The short peptide modified by the small peptide is provided to act through a blood brain barrier: the simple peptide is administered in vein or brain chamber, it is difficult to penetrate cell membrane, and it is easy to be degraded by protease in blood or tissue fluid, so it can not really function even though it obtains amino acid sequence of active small peptide with opium addiction and tolerance inhibiting effect. The invention provides a method for screening active small peptides with the functions of inhibiting opiate addiction and tolerance, which can enter cells to take effect by adding cell-penetrating peptides. The membrane penetrating peptide comprises TAT, Angiopep-2, T7 and the like, and can be respectively connected with the active small peptides to achieve the membrane penetrating effect.
3. Small peptides with active ingredients that inhibit opiate addiction and tolerance are proposed for use in medicine: mainly takes the injection mode, and is administrated through veins and ventricles. The intravenous administration is convenient, the compliance of patients is good in the future, the ventricular administration is realized, the dosage is small, the concentration of the medicine in a central system is high, and the medicine effect is possibly better. Because the cell-penetrating peptide is modified, the blood brain barrier can be penetrated by increasing the dosage through intravenous administration, and the actual use and operation of the medicine are facilitated.
4. The artificial synthesis method of the small peptide with the active ingredient for inhibiting the addiction and tolerance of the opium is provided: the active small peptide provided by the invention only has 21 amino acids, the cell-penetrating peptide only has dozens of amino acids, the total length does not exceed about 40 amino acids, and the artificial solid phase synthesis method is a relatively economic method and can realize batch production.
5. The modified short peptide can obviously improve the analgesic effect of opium, prolong the analgesic time, inhibit the tolerance of opium and correspondingly inhibit the addiction of opium: in the examples, TAT-AHD2 is shown to be capable of prolonging the opioid analgesic time remarkably, the analgesic effect of H2O and TAT-AHD3 plus morphine groups is reduced along with the prolonging of the administration time, namely morphine tolerance is realized, the TAT-AHD2 is administrated in advance to obviously inhibit the morphine tolerance, and the opioid analgesic tolerance is inhibited, so that the addiction is correspondingly inhibited.
Drawings
FIG. 1: ABIN-1 and mutant structure pattern diagram thereof
FIG. 2: immunco-precipitation detection ABIN-1 and its mutant and MOR interaction electrophoresis picture
FIG. 3: co-immunoprecipitation to verify the interaction of AHD2 with MOR
FIG. 4: co-immunoprecipitation demonstrated the interaction of AHD2 with beta-arrestin 2
FIG. 5: inhibition of MOR phosphorylation after agonist action by AHD2
FIG. 6: effect of ABIN-1 different spliceosomes on MOR phosphorylation after agonist action
FIG. 7: galaxy PepDeck (A), CABS (B) predicted complex structure
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.