WO2010066125A1

WO2010066125A1 - A short peptide, preparation and uses thereof

Info

Publication number: WO2010066125A1
Application number: PCT/CN2009/071610
Authority: WO
Inventors: 余榕捷
Original assignee: Yu Rongjie
Priority date: 2008-12-12
Filing date: 2009-04-30
Publication date: 2010-06-17
Also published as: CN101434649A; CN101434649B

Abstract

Provided are a short peptide consisting of the amino acid sequence of His-Ser-Asp-Gly-Ile and its cyclic analogue cyclo-(Cys-His-Ser-Asp-Gly-Ile-Cys). Also provided are a method for preparing the cycloheptapeptide through solid phase synthesis and uses of the short peptide and cycloheptapeptide for diagnosing or treating the diseases related to pituitary adenylate cyclase activating polypeptide (PACAP) and vasoactive intestinal peptide (VIP).

Description

Short peptide, preparation method and use thereof

[1] Technical field

[2] The present invention relates to a polypeptide having pharmacological action, in particular to a short peptide and preparation thereof

Methods and applications in the diagnosis or treatment of diseases associated with PACAP and VIP.

[3] Background Art

[4] pituitary adenylate cyclase activating polypeptide

, PACAP ) is 1989

The neuropeptides with important biological functions secreted by the pituitary gland discovered in the year belong to the new member of the secretin/glucagon/VIP family.

[5] PACAP is fairly conserved during animal evolution, and the vertebrate frog and human PACAP differ only by 2 amino acids, indicating that PACAP performs key physiological functions. PACAP and its receptors (PAC1, VPAC1 and VPAC2)

It is widely distributed in the body, not only in the central nervous system, the peripheral nervous system, but also in non-neural tissues, such as brain, testis, ovary, respiratory tract, lung, pancreas, adipose tissue, etc.

[6] PACAP has been shown to have neurotransmitters I

Regulates and protects nerve cells; promotes hormone secretion, regulates endocrine balance; regulates gonadal function and germ cell production; participates in digestive activities, regulates energy metabolism balance; participates in regulating biological functions such as immune system.

[7] There are two forms of PACAP in the body: PACAP27 and PACAP38, PACAP27 is

The N-terminus of PACAP38 is 27 amino acids. In vivo, PACAP loses its N-terminal five amino acids, His-Ser-Asp-Gly-Ile, under the action of dipeptidylpeptidase (IVDPP IV).

PACAP (6-27) and PACAP (6-38) are antagonists of PACAP. Therefore, the short peptide of the N-terminal five amino acids of PACAP (His-Ser-Asp-Gly-Ile) is maintained for PACAP.

The activity and function are very important. Currently not yet useful for linear pentapeptide His-Ser-Asp-Gly-Ile regulation Report on PACAP and VIP activity.

[8] Vasoactive intestinal peptide with high homology to PACAP ( vasoactive intestinal peptide, VIP

) also has an N-side similar to PACAP, and VIP with PACAP

It is also degraded by dipeptidase to become an antagonist of itself, and thus His-Ser-Asp-Gly-Ile also has an effect of regulating VIP activity in vivo.

[9] However, the linear pentapeptide is very unstable in vivo. On the contrary, the main chain of the cyclic peptide has a cyclic structure and has a certain conformational constraint. Its conformation has a certain stability relative to the linear peptide, and its anti-enzymatic ability is better than the linear type. Peptide is strong, so consider His-Ser-Asp-Gly-Ile

After cyclization, it improves its stability in vivo and its ability to resist enzymatic hydrolysis. There is currently no

His-Ser-Asp-Gly-Ile reports on PACAP and VIP activity after cyclization.

[10]

[11] Summary of the invention

[12] The purpose of the present invention is to provide an effective intervention for PACAP in vivo, in view of the deficiencies of the prior art.

Activity of neuropeptides such as VIP, reversing PACAP (6-27) or PACAP (6-38)

Antagonistic function to assist the body in PACAP, VIP

A short peptide with a strong conformational and anti-enzymatic ability that exerts a variety of important biological functions.

[13] Another object of the present invention is to provide a cyclic heptapeptide which is stable in the above short peptide.

Another object of the present invention is to provide a process for producing the above cyclic heptapeptide.

[15] A further object of the present invention is to provide the above short peptide or cyclic heptapeptide in diagnosis or treatment with PACAP,

Application in VIP related diseases.

[16] The above object of the present invention is achieved by the following scheme:

[17] The present invention has been extensively studied to find that the short peptide histidine-serine-aspartate-glycine-isoleucine (His-Ser-Asp-Gly-Ile) maintains APCAP, VIP

The activity and function are very important, however, the linear structure of this pentapeptide makes it very unstable in vivo. In order to overcome this problem, the present invention not only regulates by designing some amino acids or groups (such as cysteine or methyl group) at both ends of the pentapeptide, so that the resulting peptide chain is a cyclic peptide structure. APCAP, VIP

The action of the activity ensures the conformational stability of the peptide chain in the body and the strong anti-enzymatic ability. Preferably, the cysteine of the invention cyclizes the pentapeptide to effect conversion from a linear polypeptide to a cyclic peptide structure. Add cysteine Cys to both ends of His-Ser-Asp-Gly-Ile

The thiol group of the two cysteines at the beginning and the end forms an intramolecular disulfide bond, thereby producing a cyclic heptapeptide which is conformationally stable and can effectively resist the enzymatic hydrolysis of aminopeptidase and carboxypeptidase. Its structural formula is ring-(cysteine-histidine-serine

-aspartic acid-glycine-isoleucine-cysteine) [cyclo- (

Cys-His-Ser-Asp-Gly-Ile-Cys )].

[19] In the synthesis of peptides, each peptide bond needs to undergo functional steps such as functional group protection, condensation, deprotection, etc., plus the separation and purification of each step of the product, the operation is quite complicated and expensive, so now it is more effective. The synthesis of polypeptides is carried out by phase synthesis.

[20] There are two kinds of solid phase synthesis methods, namely Fmoc and tBoc, due to Fmoc ratio tBoc

There are more advantages, so most of them use the Fmoc method to synthesize peptides.

[21] The synthesis of the cyclic heptapeptide of the present invention is carried out by solid phase synthesis, preferably by the Fmoc method.

[22] The method for synthesizing the cyclic heptapeptide of the present invention comprises the following steps:

[23] (1) using a Fmoc amino acid as a raw material, and using a solid phase synthesis method to obtain a linear peptide;

[24] (2) cyclization of a linear peptide;

[25] (3

The crude product is obtained by cutting off the resin with a cutting reagent, and purified by reverse-phase high performance liquid chromatography to obtain a cyclic heptapeptide.

[26] In the above step (1), the condensing agent used in the solid phase synthesis method is TBTU/HOBT/DIEA

Complex condensing agent; TBTU : 0-(7-azobenzotriazole-1-oxo)-Ν,Ν,Ν',Ν'-tetramethylurea hexafluorophosphate; ΗΟΒΤ : Ν-hydroxybenzo Triazole; DIEA : Ν,Ν'-diisopropylethylamine; the TBTU/HOBT/DIEA complex condensing agent is Fmoc

The condensing agents commonly used in the process, the formulation and the amount thereof are well known to those skilled in the art, and the Fmoc method herein is also a general method in the art, and the operation method can be referred to relevant textbooks.

[27] In the above step (2), the cyclization of the linear peptide is carried out on the resin before shearing, such as a linear chain.

The cyclization of Cys-His-Ser-Asp-Gly-Ile-Cys is just before shearing, to Fmoc

The protected sulfhydryl group is oxidized on the resin to convert the linear pentapeptide into a cyclic heptapeptide structure; the oxidizing agent may be any oxidizing agent such as I ₂ , H ₂ 0 ₂ , Hg ² + salt, K ₃ Fe(CN) ₆ , bismuth trifluoroacetate (tfa) ₃ , chlorosilicon sulfonium sulfone oxidant or dimethyl sulfoxide DMSO. [28] Steps above (2), (3)

Linear peptide cyclization, cleavage reagent cleavage and reverse high phase liquid chromatography RP-HPLC

The steps of purification and the like are all conventional techniques in the art.

[29] Animal experiment results show that: the cycloheptapeptide of the present invention can effectively interfere with the activity of PACAP in vivo, and improve

The PACAP-mediated biological function can also complement the natural antagonists PACAP (6-27) or PACAP (6-38), reverse its antagonism, promote agonism, and enhance PACAP in vivo.

There is no similar mechanism of action on the receptor's agonistic effects and effects.

[30] Because vasoactive intestinal peptide VIP is highly homologous to PACAP and has a similar N

Therefore, the cyclic heptapeptide of the present invention also has an effect of regulating VIP activity in vivo.

[31] Therefore, the cycloheptapeptide of the present invention can be used for diagnosis and treatment with PACAP, VIP

Related diseases, due to PACAP, VIP

It is very conservative in the process of biological evolution and has a wide range of important biological functions. Therefore, the diseases which the cycloheptapeptide of the present invention may treat cover the nervous system, the endocrine system, digestion and energy metabolism, the cardiovascular cycle, the reproductive system and the immune system, such as diabetes. (including type I, II

Type), obesity, obesity-related diseases, metabolic disorders, metabolic-related syndromes, brain damage, cerebral ischemia, prevention of nerve cell death, damaged nerve cell protection and regeneration, anti-inflammatory, male impotence, sexual indifference, infertility , female sexual dysfunction, neuralgia, neuropathy, neurological disorders, anxiety, psychosis, memory impairment, dementia, cognitive confusion, central nervous system disorders, migraine, nervous contracture, myocardial ischemia, myocardial infarction /

Fibrosis, arteriosclerosis, muscle contracture, gastric ulcer, hypertension, endotoxic shock, thrombosis, retinopathy, cardiovascular disease, renal failure, heart failure, tumor cell apoptosis, tumor cell proliferation, etc.

[32] Compared with the prior art, the present invention has the following beneficial effects:

[33] 1. The cyclic heptapeptide of the present invention, including the His-Ser-Asp-Gly-Ile sequence, can specifically regulate the activity of PACAP and VIP in vivo, and can also reverse the PACAP (6-38) in vivo and The antagonistic effect of PACAP (6-27) has broad application prospects and high application value;

[34] 2. The cyclic heptapeptide of the present invention, which is used in His-Ser-Asp-Gly-Ile

Adding amino acids or groups at both ends of the sequence, such as adding cysteine at both ends, and forming a cyclic structure by using the intermolecular disulfide bond of cysteine, thereby ensuring the stability of the entire loop heptapeptide in vivo conformation, as well as Effective anti-enzymatic ability, also guarantees His-Ser-Asp-Gly-Ile

The active peptide stably functions in the body to regulate the activity of PACAP and VIP;

[35] 3.

The cyclic heptapeptide of the present invention is solid phase synthesis, and the initial reactants and products are all attached to a solid phase carrier, so that all reactions can be carried out in one reaction vessel, which is convenient for automation, and an excessive amount of reactants can be added. High yield of product, the same product is easily separated;

[36] 4. The cyclic heptapeptide of the present invention is capable of effectively compensating and reversing the pair of PACAP (6-38) and PACAP (6-27)

The antagonistic effect of PACAP, antagonism is agonistic; therefore, it can effectively interfere with the overall role and metabolic level of PACAP in vivo. There are no reports of similar mechanisms of action.

[37] 5. The cyclic heptapeptide of the present invention can be used for diagnosis and treatment of diseases associated with PACAP and VIP, in view of PACAP, VIP

It is very conservative in the process of biological evolution, and has a wide range of important biological functions. Therefore, the medical value of cycloheptapeptide is high, and the cyclic heptapeptide can be obtained by a simple solid phase synthesis method, and thus has a broad market prospect.

[38]

[39] BRIEF DESCRIPTION OF THE DRAWINGS

[40] Figure 1 is a mass spectrum of the cyclic heptapeptide CHC;

[41] Figure 2 is a HPLC detection diagram of cycloheptapeptide CHC;

[42] Figure 3 is a graph showing the effect of cyclic heptapeptide CHC on blood glucose transients;

[43] Figure 4 is a graph of blood glucose concentration of the cyclic heptapeptide CHC intervention in the glucose tolerance test;

[44] Figure 5 is a blood glucose concentration integral map of the cyclic heptapeptide CHC intervention in the glucose tolerance test;

[45] where * indicates P < 0.01;

[46] Figure 6 is a histogram of the effect of cycloheptapeptide CHC on STZ-induced diabetes;

[47] Among them, A is Buffer+ saline, B is STZ+ saline, C is STZ+PACAP38,

D is STZ+HSDGI, E is STZ+cycloheptapeptide CHC, △ means Ρ<0·01 (Β group vs. group A), * means Ρ<0·01 (group D vs. group B, group v vs. B) Group);

[48] Figure 7 is a bar graph of the reversal of PACAP (6-38) antagonistic nerve growth by cycloheptide CHC;

[49] Among them, A is a blank control, B is PACAP38, C is PACAP (6-38), and D is CHC ten. PACAP (6-38), E is PACAP (6-38) + PACAP38, F is CHC+ PACAP (6-38) + PACAP38, △ means Ρ<0·01 (group B vs. E group), * means Ρ< 0·01 (Group D vs. Group C), ** means Ρ<0·01 (Group F vs. E).

[50] Detailed Description

[51] Example 1 Preparation of cycloheptapeptide (CHC)

[52] The cyclic heptapeptide of the present example is prepared by solid phase synthesis, and the specific steps are as follows:

[53] (1) Synthesis of linear heptapeptide by conventional Fmoc solid phase synthesis: with Fmoc

Amino acid as raw material, solid phase synthesis, TBTU/HOBT/DIEA

Synthesis of linear peptide Cys-His- Ser-Asp-Gly-Ile-Cys catalyzed by complex condensing agent;

[54] ( 2 ) Solid phase cyclization: After the last amino acid is taken, the last amino acid Cys is not removed.

Fmoc protecting group, the cyclization solution containing an oxidizing agent is added. 1 and ₂ (weighed 2.6 gl ₂ was dissolved in 85 mL of methanol and 15 mL of dimethylformamide DMF mixture, dubbed 0.1 mol / L 1 ₂ solution),

Mix and add to the reaction vessel, blow nitrogen for 6 h, oxidize a SH to form disulfide bond-S-S

Loop into

[55] ( 3 ) Add trifluoroacetic acid TFA / thioanisole / mercaptoethanol / water in a ratio of 10 mL / 0.5 mL / 0.25 mL / 0.5 mL / 0.75 g /

a cleavage reagent for phenol preparation, which is cleaved from the resin to obtain a crude product, and the crude product is purified by RP-HPLC to obtain a cycloheptapeptide [cyclo-(Cy-His-Ser-Asp-Gly-Ile-Cys)]. Referred to as CHC

[56] The above-mentioned cyclic heptapeptide was subjected to flight mass spectrometry MALDI-MS.

[57] The cyclic heptapeptide of the present embodiment is solid phase synthesis, which avoids the production of liquid phase cyclized dimers or multimers, and has a high reaction concentration and low cost. The molecular weight of the cyclic heptapeptide of this example is 732.3.

The yield can reach 19%, and the purity after purification is over 96%. The mass spectrometry and HPLC detection of the synthesized cyclic heptapeptide are shown in Fig. 1 and Fig. 2.

[58] Example 2 Effect of cycloheptapeptide (CHC) on blood glucose

[59] Clean grade (SPF grade) NIH male mice (use license number: Guangdong inspection certificate 2002-2009; Certificate number: Guangdong inspection certificate 2003A 076

), provided by the Experimental Animal Center of the First Military Medical University, weighing 25 ± 5g, randomly divided into 2 by weight Group, 10 rats in each group were injected intraperitoneally with 500 μg/kg CHC cycloheptapeptide and normal saline (0.9% by mass NaCl) at Omin, lOmin, 20min, 30 min, 45 min and 60 min.

The blood glucose was measured, and the results are shown in Fig. 3. The CHC cycloheptapeptide has an activity of regulating blood sugar transiently, which is consistent with the activity of PACAP.

[60] Example 3 Modulation of glucose tolerance by cycloheptapeptide (CHC)

[61] Clean grade (SPF grade) NIH male mice, supplied by the Experimental Animal Center of the First Military Medical University, weighed 25 ± 5 g, randomized by body weight, 10 in each group. Weighing, numbering, fasting 18h

, by intraperitoneal injection of 1.8mmol glucose / kg ten 500 μ g / kg cycloheptapeptide CHC, with 1.8mmol glucose / kg ten saline (Saline) as a control, blood glucose concentration at Omin, 15min, 30min, 45min and 60min The blood glucose concentration curve is shown in Figure 4. Figure 5 is the blood glucose concentration integral map of the CHC cycloheptapeptide intervention in the glucose tolerance test. It can be seen from Figure 4 and Figure 5 that the cyclic heptapeptide CHC significantly promotes glucose tolerance. Role, CHC

The cycloheptapeptide can effectively improve the body's ability to remove high glucose (P<0.01).

[62] Example 4 Intervention of cycloheptapeptide (CHC) in STZ-induced diabetic mice

[63] Clean-grade Kunming male mice weighing 26-30 g, adaptive feeding for 5 d, fasting for 16 h,

Tail vein blood sampling is used to measure fasting blood glucose with a blood glucose meter. The blood glucose level is less than 6 mol/L.

The mice entered the trial. Randomly divided into 5 groups according to body weight, 10 mice in each group, fasted for 16h

After the intraperitoneal injection of the following five groups of drugs:

[64] Group A: citric acid buffer (Buffer) ten physiological saline (0.9% by mass NaCl);

[65] Group B: 50 mg/kg streptozotocin (STZ) ten normal saline (0.9 mass% NaCl); group C: 50 mg/kg STZ ten 500 μg/kg PACAP38;

[66] Group D: 50 mg/kg STZ ten 500 μg/kg HSDGI linear pentapeptide (HSDGI linear pentapeptide is His-Ser-Asp-Gly-Ile linear peptide);

[67] Group E: 50 mg/kg STZ ten 500 μg/kg CHC cycloheptapeptide.

[68] Five days after continuous injection, fasting blood glucose was measured for each group after 7 days.

[69] The results are shown in Figure 6: STZ compared with the blank control group injected with Buffer ten saline

The blood glucose level in the 10 saline group was significantly increased (P<0.01), indicating that the STZ induced diabetes model was successful; the blood glucose rise in the STZ ten PACAP38 group was slightly inhibited, but not statistically significant; STZ ten CHC Both the cycloheptapeptide and the STZ-10 HSDGI group were effective (P<0.01), which inhibited the rise of blood glucose, indicating that CHC cycloheptapeptide and HSDGI linear peptide are resistant to STZ damage to islet cells, and CHC loop heptapeptide ratio

HSDGI linear peptides work better.

[70] Example 5 Cycloheptapeptide (CHC) reversal of PACAP (6-38) antagonizing nerve growth

[71] Culture of PC 12 cells (PC-12 cells are a commonly used nerve cell line, commercially available), and the culture medium contains

5 % newborn calf serum and 10% horse serum in DMEM, 37. C. The volume fraction is 0.05 C0 _{2 in the} incubator.

[72] 1 xl07 / L of PC 12 cells were seeded in 96-well plastic plates coated with poly-L-lysine poly-L lysine, 100 μM per well. After the cells were attached, the cells were randomly divided into cells. 6 groups:

[73] Α : blank control;

[74] Β: PACAP38;

[75] C, PACAP (6-38);

[76] D, CHC 10 PACAP (6-38);

[77] Ε, PACAP (6-38) + PACAP38;

[78] F, CHC+ PACAP (6-38) + PACAP38.

[79] Cultured for 72 h, observed cell growth. If the length of the cell protrusion is greater than or equal to 1 times the diameter of the cell body, it is a positive growth cell. Random number 150~200 cells,

Calculate the percentage of positive growth cells. Each set of data comes from two experiments.

[80] The results are shown in Fig. 7. PACAP(6-38) itself has no obvious effect on the culture of nerve cells in vitro, but CHC10PACAP(6-38) effectively promotes the growth of PC12 (*ρ<0·01). A similar effect to PACAP38 suggests that CHC can effectively compensate for the function of PACAP (6-38).

[81] While PACAP (6-38) itself inhibits the action of PACAP38 on neurite outgrowth (△ p<0.01

However, after the addition of CHC, the inhibitory effect was converted into a promoting effect. The CHC+PACAP(6-38) ten PACAP38 group had a higher percentage of neurites than the PACAP38 group (**ρ<0·01).

[82] The above results indicate that the CHC cycloheptapeptide has an effective function of compensating and reversing the action of PACAP (6-38).

Claims

Claim

[1] A short peptide whose amino acid sequence is histidine-serine-aspartate-glycine-isoleucine.

[2] A cyclic heptapeptide containing the short peptide of claim 1, which has the structural formula of cyclo-(cysteine-histidine-serine-aspartate-glycine-isoleucine-cysteine Acid).

[3] The cyclic heptapeptide according to claim 2, wherein the cyclic heptapeptide is cyclized in such a manner that a disulfide bond is formed between the cysteine at the 1st position and the cysteine at the 7th position, thereby becoming a Ring structure.

[4] A preparation claim 2

The method for the cycloheptapeptide, characterized in that the method is a solid phase synthesis method.

[5] Claim 1

The use of the short peptide for diagnosing or treating a disease associated with pituitary adenylate cyclase activating polypeptide, vasoactive intestinal peptide.

[6] Claim 2

The use of the cyclic heptapeptide in the diagnosis or treatment of a disease associated with pituitary adenylate cyclase activating polypeptide, vasoactive intestinal peptide.

[7] The use according to any one of claims 5 or 6, characterized in that the disease is

Diabetes, obesity, obesity-related diseases, metabolic disorders, metabolic-related syndromes, brain damage, cerebral ischemia, prevention of nerve cell death, damaged nerve cell protection and regeneration, anti-inflammatory, male impotence, sexual indifference, infertility, Female sexual dysfunction, neuralgia, neuropathy, neurological disorders, anxiety, psychosis, memory impairment, dementia, cognitive confusion, central nervous system diseases, migraine, neurological contraction, myocardial ischemia, myocardial infarction, myocardial

Fibrosis, arteriosclerosis, muscle contracture, gastric ulcer, hypertension, endotoxic shock, thrombosis, retinopathy, cardiovascular disease, renal failure, heart failure, tumor cell apoptosis, or tumor cell proliferation.