Novel somatotropin-releasing hormone analogous peptide modification and dimerization preparation and application thereof

文档序号：1373580 发布日期：2020-08-14 浏览：19次中文

阅读说明：本技术 新型生长激素释放激素类似肽改构和二聚体化制备及其应用 (Novel somatotropin-releasing hormone analogous peptide modification and dimerization preparation and application thereof ) 是由唐松山张旭东杨莉谭宏梅罗群唐婧晅于 2020-03-18 设计创作，主要内容包括：本发明提供了人生长激素释放激素(hGHRH)类似肽的分子变构体和其二聚体在治疗GH缺乏症、不孕不育症、老年痴呆症、糖尿病和增强免疫力中的应用。本发明二聚体是两个相同含有单个半胱氨酸分子变构的hGHRH类似肽单体通过半胱氨酸形成的二硫键连接而成,并形成H型结构(分子内部单Ser→Cys替换)。本发明还对GHRH类似肽的其中一个赖氨酸的侧链ε-氨基进行脂肪酸链修饰。通过脂肪酸修饰延长GHRH类似肽单体或二聚体在体内GH持续释放时间,最长达17天。本发明发现,具有长效活性的GHRH二聚体具有Aib→2Ala代换、18C-18C二硫键形成、C20脂肪酸链修饰以及C端酰胺化结构。(The invention provides the application of molecular variant of human growth hormone releasing hormone (hGHRH) peptide and dimer thereof in treating GH deficiency, infertility, senile dementia, diabetes and enhancing immunity. The dimer of the invention is formed by two same hGHRH-like peptide monomers containing single cysteine molecule allosteric through cysteineDisulfide bonds, and forms an H-type structure (single Ser → Cys substitution within the molecule). The invention also carries out fatty acid chain modification on the side chain-amino group of one lysine of the GHRH analog peptide. The GH sustained release time of the GHRH-like peptide monomer or dimer in vivo is prolonged by fatty acid modification, and reaches 17 days at most. The invention discovers that GHRH dimer with long-acting activity has Aib → 2 Ala substitution, 18 C‑ 18 C disulfide bond formation, C20 fatty acid chain modification, and C-terminal amidation structures.)

1. A Growth Hormone Releasing Hormone (GHRH) -like peptide monomer, wherein serine S on the peptide chain is substituted with cysteine C, or threonine T is substituted with homocysteine (Hcy), and one of the lysines on the main peptide chain is a lysine that is side chain-amino modified with a glutamyl fatty acid [ γ -Glu (N- α -fatty acid) ] or glutamyl fatty diacid [ γ -Glu (N- α -fattydiacide) ] or [2 × AEEAC- γ -Glu (N- α -fatty acid) ].

2. The GHRH analog peptide monomer of claim 1, wherein the analog peptide monomer has a specific sequence selected from any one of:

(1)(NH₂)X₁-X₂-DAIFTNCY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMSR-NH₂or;

(2)(NH₂)X₁-X₂-DAIFTNSY-X₁₁-X₁₂-VLGQLCA-X₂₀-X₂₁-LLQDIMSR-NH₂or;

(3)(NH₂)X₁-X₂-DAIFTNSY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMCR-NH₂or;

(4)(NH₂)X₁-X₂-DAIF-Hcy-NSY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMS-X₂₉-NH₂or;

(5)(NH₂)X₁-X₂-DAIFTNCY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMS-X₂₉-QQGESNQE-X₃₈-GA-X₄₁-A-X₄₃-L-NH₂or;

(6)(NH₂)X₁-X₂-DAIFTNSY-X₁₁-X₁₂-VLGQLCA-X₂₀-X₂₁-LLQDIMS-X₂₉-QQGESNQE-X₃₈-GA-X₄₁-A-X₄₃-L-NH₂or;

(7)(NH₂)X₁-X₂-DAIFTNSY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMC-X₂₉-QQGESNQE-X₃₈-GA-X₄₁-A-X₄₃-L-NH₂or;

(8)(NH₂)X₁-X₂-DAIFTNSY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMS-X₂₉-QQGECNQE-X₃₈-GA-X₄₁-A-X₄₃-L-NH₂or;

(9)(NH₂)X₁-X₂-DAIF-Hcy-NSY-X₁₁-X₁₂-VLGQLSA-X₂₀-X₂₁-LLQDIMS-X₂₉-QQGESNQE-X₃₈-GA-X₄₁-A-X₄₃-L-NH₂

wherein, X₁Is P or Y or F; x₂Is A or α -aminoisobutyric acid (α Aib); X₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is K, or is R, or is a side chain-amino glutamyl fatty acid [ gamma-Glu (N- α -fat acid)]Or glutamyl fatty diacid gamma-Glu (N- α -fatty diacid) modified lysine, or side chain-amino [2 × AEEAC-gamma-Glu (N- α -fatty diacid)]A modified lysine.

3. The somatotropin releasing hormone analog peptide monomer of claim 2 wherein when X is₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is side chain-amino glutamyl fatty acid [ gamma-Glu (N- α -fat acid)]Or glutamyl fatty diacid gamma-Glu (N- α -fat diacid) modified lysine, the structure is shown in chemical formula 1;when X is present₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is on the side chain-amino group [2 × AEEAC-gamma-Glu (N- α -fatty diacid)]Modified lysine, the structure of which is shown in chemical formula 2:

chemical formula 1:

chemical formula 2:

4. a GHRH-like peptide homodimer formed by the linkage of identical monomers according to any one of claims 1 to 3 via a cysteine-forming disulfide bond, constituting a type H GHRH-like peptide homodimer.

5. The dimer of claim 4, wherein the amino acid sequence is any one of:

6. The somatotropin releasing hormone analog peptide monomer of claim 5 wherein when X is₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is side chain-amino glutamyl fatty acid [ gamma-Glu (N- α -fat acid)]Or glutamyl fatty diacid gamma-Glu (N- α -fatty diacid) modified lysine, the structure is shown in chemical formula 1, when X is₁₁Or X₁₂Or X₂₀Or X₂₁Or X₂₉Or X₃₈Or X₄₁Or X₄₃Is on the side chain-amino group [2 × AEEAC-gamma-Glu (N- α -fatty diacid)]The structure of modified lysine is shown in chemical formula 2.

7. Use of a ghrelin-like peptide according to any of claims 1 to 3, or a homodimer according to any of claims 4 to 6, for the manufacture of a medicament for the treatment of GH deficiency, infertility, senile dementia, immune enhancement or diabetes.

8. A medicament for treating GH deficiency, infertility, senile dementia, immune enhancement or diabetes, which comprises the ghrelin-like peptide of any one of claims 1 to 3, or the homodimer of any one of claims 4 to 6 and a pharmaceutically acceptable salt thereof as an active ingredient.

Technical Field

The invention belongs to the field of medical biology, and particularly relates to preparation of various human novel growth hormone releasing hormone similar peptide monomers and dimers and application thereof in treatment.

Background

Central growth hormone-releasing hormone-growth hormone-insulin-like growth factor (GHRH-GH-IGFs) endocrine axis, regulate body cell growth and sexual cell reproduction. The human GHRH complete sequence is hGHRH (1-44) NH₂hGHRH (1-29) is a core peptide with 51% bioactivity. The in vitro or in vivo activity of the novel hGHRH-like peptides and dimers thereof can be determined by measuring Growth Hormone (GH) release values by pituitary incubation in vitro or subcutaneous injection in vivo.

New studies have found that the effect of hGHRH molecules on peripheral tissues differs from that on central mechanisms of action, such as in the central, hypothalamic GHRH-pituitary GHRH receptor-pituitary GH with axis-specific effects, in the ovaries or testis of mature animals, although they also contain GHRH, GHRH receptor and GH molecular signals, they do not necessarily produce such axis-specific effects. In vivo injection of some novel GHRH analog peptides or dimeric molecules thereof will produce up-regulation of the GHRH receptor in germ cells to increase conception rates without promoting increased GH in vivo, indicating that reproduction of mature animals in subjects is not directly related to GH. In vitro pituitary incubation of such peptides (which do not produce GH in vivo) induces pituitary GH secretion. Therefore, the reproduction relationship of GH and mature animals is shown by measuring GH in vivo and in vitro pituitary GH secretion and the pregnancy rate of representative GHRH test animals of the test animals, and the new possibility of the peptides in treatment is shown.

GHRH peptide has wide pharmacological activity, such as treating infertility, regulating immunity, senile dementia, promoting wound healing, reconstructing myocardial cell, improving sleep quality, reducing weight or treating diabetes etc.

Although hGHRH analogs have great potential for use in biomedical and agronomic fields, their short half-life or low activity limits their utility. The structure-activity relationship indicates that the N-terminus¹Y is required for pituitary GH release in GHRH.¹P-GHRH shows pituitary GH release in vitro, but no GH release in vivo.¹F-GHRH shows very low GH release in vitro and in vivo. As the sequence extends towards the C-terminus, its activity and half-life increase. When two amino acid residues at the N-terminal of the GHRH molecule are cleaved by aminopeptidase (DPP-4) in the blood, the molecule is inactivated. hGHRH (1-29) NH₂Has a half-life of 13 minutes, natural hGHRH (1-44) NH₂Half life 17 minutes. Up to now, hGHRH (1-44) NH₂Is the most active of these analogs.

Many protein precursors need to be associated with long fatty chains in the endoplasmic reticulum to increase maturation processing ability, or some protein molecules distributed in the cell membrane, the endoplasmic reticulum membrane or the golgi membrane, etc. will be anchored to these membrane phase systems via long fatty acid chains to increase localization ability. These fatty acylated GHRH molecules significantly increase half-life and at the same time, multiply increase their molecular activity. The invention utilizes the technologies to generate a series of novel long-acting GHRH molecules, and is applied to the treatment of infertility, immunity regulation, senile dementia, wound healing promotion, myocardial cell reconstruction, sleep quality improvement, weight reduction or diabetes.

Disclosure of Invention

It is an object of the present invention to overcome the above-mentioned deficiencies of the prior art and to provide Growth Hormone Releasing Hormone (GHRH) like peptide monomers. The GHRH-like peptide of the present invention has a molecular allosteric conformation to the main peptide chain, on the one hand, a single C → S or homocysteine (Hcy) → T substitution inside the monomeric peptide, and the position of cysteine in the peptide chain has been studied. On the other hand, the side chain-amino group of lysine (K) in the peptide chain is modified with a glutamyl fatty acid or glutamyl fatty diacid or [2 × AEEAC- γ -Glu (N- α -fatty diacid) ] substituent.

In order to achieve the purpose, the invention adopts the technical scheme that: a Growth Hormone Releasing Hormone (GHRH) -like peptide monomer, wherein a serine S of the peptide chain is substituted with a cysteine C, or a threonine T is substituted with a homocysteine (Hcy), and one of the lysines of the main peptide chain is a lysine modified on the side chain-amino group with a glutamyl fatty acid [ γ -Glu (N- α -fatty acid) ] or glutamyl fatty diacid [ γ -Glu (N- α -fatty diacid) ] or [2 × AEEAC- γ -Glu (N- α -fatty diacid) ].

Preferably, the specific sequence of the analogous peptide monomer is any one of the following: