CN101434649A

CN101434649A - Small peptide, preparation thereof and use thereof in diagnosis or treatment of PACAP and VIP related diseases

Info

Publication number: CN101434649A
Application number: CNA2008102198844A
Authority: CN
Inventors: 余榕捷
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-12-12
Filing date: 2008-12-12
Publication date: 2009-05-20
Anticipated expiration: 2028-12-12
Also published as: WO2010066125A1; CN101434649B

Abstract

The invention discloses a short peptide, a preparation method thereof and the application in the diagnoses and cure of diseases related to PACAP and VIP. The sequence of the short peptide is histidine-serine-aspartic acid-glycin-isoleucine; by adding two cysteines at the head and the end of the short peptide, the sulphydryls of the two cysteines forms disulfide bonds and further a circular 7-mer peptide is formed by cyclization. The circular 7-mer peptide prepared by the method comprises the His-Ser-Asp-Gly-Ile sequence, can specifically regulate the activation of PACAP and VIP in a body, and can reverse the antagonistic effect of PACAP(6-38) and PACAP(6-27) in the body simultaneously, thereby having wide application prospect and higher application value. The circular 7-mer peptide can be obtained by a solid phase synthetic method which is easy to be operated, thereby having very broad marketing prospect.

Description

A kind of small peptide and preparation method thereof and the application in diagnosis or treatment and PACAP, VIP relative disease

Technical field

The present invention relates to have the polypeptide of pharmacological action, be specifically related to a kind of small peptide and preparation method thereof and the application in diagnosis or treatment and PACAP, VIP relative disease.

Background technology

Pituitary adenylate cyclase-activating polypeptide. (pituitary adenylate cyclase activatingpolypeptide, PACAP) be discovery in 1989, by the nerve polypeptide that important biomolecule is learned function that has of pituitary secretion, to belong to be secretin/glucagon/ newcomer in the VIP family.

PACAP is quite conservative in the zoogeny process, and vertebrates frog and people's PACAP has only 2 amino acid whose differences, shows that PACAP carries out crucial physiologic function.PACAP and acceptor thereof (PAC1, VPAC1 and VPAC2) are in body distribution and extensive, not only be distributed in central nervous system, peripheral nervous system, and in non-nervous tissue, also extensively exist, as brain, testis, ovary, respiratory tract, lung, pancreas, fatty tissue etc.

Confirmed that at present PACAP has neurotransmitter/modified, neuroprotective cell; Promote hormone secretion, the endocrine regulation balance; Regulate the generation of gonad function and sexual cell; Participate in the digestion activity, regulate the energy metabolism balance; Participate in regulating biological functions such as immunity system.

Intravital PACAP has two kinds of form: PACAP27 and PACAP38, and PACAP27 is 27 amino acid of N end of PACAP38.PACAP loses the five amino acid of N end in the body under the effect of two basic peptases (dipeptidylpeptidase, IVDPP IV)---and PACAP (6-27) and PACAP (6-38) that His-Ser-Asp-Gly-Ile forms they are the antagonists of PACAP.Therefore the small peptide (His-Ser-Asp-Gly-Ile) of the N of PACAP end five amino acid composition is very important for the activity of keeping PACAP and function.Not useful as yet at present linear pentapeptide His-Ser-Asp-Gly-Ile regulation and control PACAP and the active report of VIP.

With PACAP vasoactive intestinal peptide (the vasoactive intestinal peptide of high homology is arranged, VIP) also have the N end similar with PACAP, and thereby VIP becomes self antagonist with the same degraded that is subjected to two basic peptases of PACAP, so His-Ser-Asp-Gly-Ile also has the active effect of VIP in the regulation and control body.

Yet linear pentapeptide is very unstable in vivo, opposite cyclic peptide main chain is structure in the form of a ring, has certain conformation effect of contraction, the relative line style peptide of its conformation has certain stability, its resistance to enzymolysis energy force rate line style peptide is strong, so can consider to improve its stability and resistance to enzymolysis ability in vivo after the His-Ser-Asp-Gly-Ile cyclisation.Do not have as yet at present regulation and control PACAP and the active report of VIP after the His-Ser-Asp-Gly-Ile cyclisation.

Summary of the invention

The objective of the invention is at the deficiencies in the prior art, a kind of activity that can effectively intervene neuropeptides such as PACAP, VIP in the body is provided, reverse the antagonism function of PACAP (6-27) or PACAP (6-38), thereby assist intravital PACAP, VIP to bring into play multiple important biomolecule and learn function, the conformation stable and strong small peptide of resistance to enzymolysis ability.

Another object of the present invention provides the ring seven peptide of above-mentioned small peptide being made good stability.

Another object of the present invention is to provide the preparation method of above-mentioned ring seven peptide.

Further purpose of the present invention is to provide the application in diagnosis or the treatment disease relevant with PACAP, VIP of above-mentioned small peptide or ring seven peptide.

Above-mentioned purpose of the present invention is achieved by following scheme:

The present invention is through a large amount of discovering, small peptide Histidine-Serine-aspartic acid-glycine-Isoleucine (His-Ser-Asp-Gly-Ile) is very important for the activity of keeping APCAP, VIP and function, yet the linear structure of this pentapeptide makes it very unstable in vivo.In order to overcome this problem, the present invention is by designing some amino acid or group (as halfcystine or methyl) at the two ends of this pentapeptide, make that the peptide chain after generating is a cyclic peptide structures, then not only can play the active effect of APCAP, VIP of regulating, can guarantee that again peptide chain conformation in vivo is stable, and stronger resistance to enzymolysis ability.

The preferred halfcystine of the present invention carries out cyclisation to pentapeptide, realizes the conversion from the linear polypeptide to the cyclic peptide structures.Halfcystine Cys is added at two ends at His-Ser-Asp-Gly-Ile respectively, and the sulfydryl of two halfcystines of head and the tail forms intramolecular disulfide bond, thereby generates the ring seven peptide that aminopeptidase and carboxypeptidase enzymolysis are stablized and be highly resistant to conformation.Its structural formula is ring-(halfcystine-Histidine-Serine-aspartic acid-glycine-Isoleucine-halfcystine) [cyclo-(Cys-His-Ser-Asp-Gly-Ile-Cys)].

In polypeptide was synthetic, peptide bond of every generation all needed through protective group, condensation, goes operation steps such as protection, adds the separation and purification of each step product, and operation is quite complicated and time-consuming, and therefore many now employing solid-phase synthesis carry out synthesizing of polypeptide.

Solid-phase synthesis has two kinds, i.e. Fmoc and tBoc more many advantage because Fmoc exists than tBoc, so adopt the Fmoc method to synthesize polypeptide mostly again.

The synthetic employing solid-phase synthesis of ring seven peptide of the present invention, preferred Fmoc method.

The synthetic method of ring seven peptide of the present invention comprises the steps:

(1) is raw material with Fmoc amino acid, adopts solid-phase synthesis, obtain linear peptides;

(2) cyclisation of linear peptides;

(3) break away from resin with the cutting reagent cutting and obtain thick product, the RPLC purifying obtains ring seven peptide.

In the above-mentioned steps (1), the condensing agent that solid-phase synthesis adopted is the agent of TBTU/HOBT/DIEA coupling condenser; TBTU:O-(7-azo benzotriazole-1-oxygen)-N, N, N ', N '-tetramethyl-urea phosphofluoric acid ester; The HOBT:N-hydroxybenzotriazole; DIEA:N, N '-diisopropylethylamine; This TBTU/HOBT/DIEA coupling condenser agent is a condensing agent commonly used in the Fmoc method, and its prescription and consumption are that those skilled in the art know altogether, and Fmoc method herein also is the universal method of this area, and its working method can be with reference to relevant textbook.

In the above-mentioned steps (2), the cyclisation of linear peptides is carried out on resin before shearing, cyclisation as straight chain C ys-His-Ser-Asp-Gly-Ile-Cys is exactly before shearing, the sulfydryl of Fmoc protection is carried out oxidation on the resin, thereby linear pentapeptide is transformed into the ring seven peptide structure; Oxygenant can be for any one oxygenant, as I ₂, H ₂O ₂, Hg ²⁺Salt, K ₃Fe (CN) ₆, thallium trifluoroacetate T ₁(tfa) ₃, chlorosilane-sulfone class oxygenant or dimethyl sulfoxide (DMSO) DMSO etc.

Above-mentioned steps (2), the linear peptides cyclisation in (3), steps such as cutting reagent cutting and reverse high phase liquid chromatography RP-HPLC purifying are the ordinary skill in the art.

Experimentation on animals is the result show: ring seven peptide of the present invention can effectively be intervened the activity of PACAP in the body, improve the biological function of PACAP mediation, can also with natural agonist PACAP (6-27) or PACAP (6-38) complementation, reverse its antagonistic action, promote agonism, improve exciting power and the action effective of the interior PACAP of body, also do not have the report of similar effect mechanism at present acceptor.

Because vasoactive intestinal peptide VIP and PACAP have high homology, and has similar N end, so ring seven peptide of the present invention also has the active effect of VIP in the regulation and control body.

Therefore; ring seven peptide of the present invention can be used for diagnosis and treatment and PACAP; the disease that VIP is relevant; because PACAP; VIP is very conservative in the organic evolution process; has extensive and important biological function; so the disease that ring seven peptide of the present invention may be treated covers neural system; endocrine system; digestion and energy metabolism; cardiovascular circulation; reproductive system and immunity system; (comprise the I type as diabetes; the II type); fat; obesity-related disease; metabolic disturbance; the metabolism syndromes of being correlated with; brain injury; cerebral ischemia; prevent nerve cell death; injured nerve cytoprotective and regeneration; anti-inflammatory; impotence; hyposexuality; infertile; Female sexual dysfunction; neurodynia; neuropathy; dizzy; anxiety disorder; psychosis, memory impairment; dull-witted; Cog Disorg; central nervous system disease; migraine; nerve is shunk; myocardial ischemia; myocardial infarction/fibrosis; arteriosclerosis; the flesh disease of shrinking; stomach ulcer; hypertension; interior toxicogenic shock; thrombosis; retinopathy; cardiovascular disorder; renal failure; heart failure; apoptosis of tumor cells; tumor cell proliferation etc.

Compared with prior art, the present invention has following beneficial effect:

1. ring seven peptide of the present invention, comprise the His-Ser-Asp-Gly-Ile sequence, thereby the activity of PACAP, VIP in the control agent specifically, the antagonistic action of intravital PACAP of also reversible commentaries on classics (6-38) and PACAP (6-27) has broad application prospects and higher using value simultaneously;

2. ring seven peptide of the present invention, employing is added amino acid or group at the two ends of His-Ser-Asp-Gly-Ile sequence, as add halfcystine respectively at two ends, utilize the intermolecular disulfide bond of halfcystine to form ring texture, thereby guaranteed whole ring seven peptide stablizing of conformation in vivo, and effective resistance to enzymolysis ability, guaranteed that also the His-Ser-Asp-Gly-Ile bioactive peptide plays consistently the active function that it regulates PACAP, VIP in vivo;

3. ring seven peptide of the present invention adopts solid-phase synthesis, initial reaction thing and product all are to be connected on the solid phase carrier, therefore can carry out all reactions in a reaction vessel, are convenient to automated operation, the adding excessive reactant can obtain the product of high yield, and product is easy to separate simultaneously;

4. ring seven peptide of the present invention can effectively compensate and reverse PACAP (6-38) and PACAP (6-27) antagonistic action to PACAP, and the change antagonistic action is agonism; Therefore mass action and the metaboilic level that can effectively intervene PACAP in the body.The report that does not also have at present similar effect mechanism.

5. ring seven peptide of the present invention can be used in diagnosis and the treatment disease relevant with PACAP, VIP, in view of PACAP, VIP very conservative in the organic evolution process, and has an extensive and important biological function, so medical value height of ring seven peptide, and this ring seven peptide promptly can obtain by solid-phase synthesis simple to operate, therefore has very vast market prospect.

Description of drawings

Fig. 1 is the mass spectrum of ring seven peptide CHC;

Fig. 2 is that the HPLC of ring seven peptide CHC detects figure;

Fig. 3 is the instantaneous influence curve figure of ring seven peptide CHC to blood sugar;

Fig. 4 intervenes the blood sugar concentration graphic representation of sugar tolerance experiment for ring seven peptide CHC;

Fig. 5 intervenes the blood sugar concentration integrogram of sugar tolerance experiment for ring seven peptide CHC;

Wherein, ^*Expression P＜0.01;

Fig. 6 is the intervention effect histogram of ring seven peptide CHC to STZ inductive diabetes;

Wherein, A is a Buffer+ physiological saline, and B is a STZ+ physiological saline, and C is STZ+PACAP38, and D is STZ+HSDGI, and E is STZ+ ring seven peptide CHC, and △ represents P＜0.01 (B group vs.A group), ^*Expression P＜0.01 (D group vs.B group, E group vs.B group);

Fig. 7 reverses the histogram of PACAP (6-38) antagonism nerve growth effect for ring seven peptide CHC;

Wherein, A is a blank, and B is PACAP38, and C is PACAP (6-38), and D is CHC+PACAP (6-38), and E is PACAP (6-38)+PACAP38, and F is CHC+PACAP (6-38)+PACAP38, and △ represents P＜0.01 (B group vs.E group), ^*Expression P＜0.01 (D group vs.C group), ^*Expression P＜0.01 (F group vs.E group).

Embodiment

The preparation of embodiment 1 ring seven peptide (CHC)

The ring seven peptide of present embodiment adopts the solid-phase synthesis preparation, and its concrete steps are as follows:

(1) Fmoc solid-phase synthesis synthesizing linear seven peptides routinely: with Fmoc amino acid is raw material, adopts solid-phase synthesis, through the synthetic linear peptides Cys-His-Ser-Asp-Gly-Ile-Cys of TBTU/HOBT/DIEA coupling condenser agent catalysis;

(2) solid phase cyclization: after having connect last amino acid, wouldn't remove the Fmoc blocking group of last amino acid Cys, add and contain oxygenant I ₂Cyclisation solution (take by weighing 2.6g I ₂Be dissolved in 85mL methyl alcohol and the 15mL dimethyl formamide DMF mixed solution, be made into 0.1mol/L I ₂Solution), mixing adds in the reaction vessel, nitrogen blowing reaction 6h, and oxidation-SH generation disulfide linkage-S-S-, Cheng Huan;

(3) ratio in 10mL/0.5mL/0.25mL/0.5mL/0.75g adds the trifluoroacetic acid TFA/ cutting reagent that thioanisole/mercaptoethanol/water/phenol is made into, break away from resin with this cutting reagent cutting and obtain thick product, obtain ring seven peptide [cyclo-(Cys-His-Ser-Asp-Gly-Ile-Cys)] with this thick product of RP-HPLC purifying, be called for short CHC.

Above-mentioned ring seven peptide is carried out flight mass spectrum MALDI-MS to be detected.

The ring seven peptide of present embodiment adopts solid-phase synthesis, has avoided the dimer or the polymeric generation of liquid phase cyclisation, and the reaction density height, and cost is low.The molecular weight of present embodiment ring seven peptide is 732.3, and productive rate can reach 19%, and purity reaches more than 96% behind the purifying, and the mass spectrum of synthetic gained ring seven peptide and HPLC detect as shown in Figure 1 and Figure 2.

Embodiment 2 ring seven peptides (CHC) are to the instantaneous influence of blood sugar

Cleaning level (SPF level) NIH male mice (occupancy permit number: Guangdong probatio inspectionem pecuoarem word 2002-2009; Conformity certification number: Guangdong probatio inspectionem pecuoarem word 2003A076), provide body weight 25 ± 5g by No.1 Military Medical Univ.'s Experimental Animal Center, divide 2 groups at random by body weight, 10 every group, difference abdominal injection 500 μ g/kg CHC ring seven peptides and physiological saline (0.9 quality % NaCl), at 0min, 10min, 20min, 30min, 45min and 60min measure blood sugar, the result as shown in Figure 3, the CHC ring seven peptide has the activity of instantaneous blood sugar regulation and the activity of PACAP matches.

Embodiment 3 ring seven peptides (CHC) are to the adjusting of sugar tolerance

Cleaning level (SPF level) NIH male mice is provided by No.1 Military Medical Univ.'s Experimental Animal Center, body weight 25 ± 5g, and by the body weight random packet, 10 every group.Weigh, numbering, fasting 18h, by body weight abdominal injection 1.8mmol glucose/kg+500 μ g/kg ring seven peptide CHC, so that 1.8mmol glucose/kg+ physiological saline (Saline) in contrast, in 0min, 15min, 30min, 45min and 60min measure blood sugar concentration, the blood sugar concentration graphic representation as shown in Figure 4, Fig. 5 intervenes the blood sugar concentration integrogram that sugar tolerance is tested for the CHC ring seven peptide simultaneously, by Fig. 4 and Fig. 5 as can be seen ring seven peptide CHC sugar tolerance is had significant promoter action, the CHC ring seven peptide can effectively improve the ability (P＜0.01) that body is removed high sugar.

Embodiment 4 ring seven peptides (CHC) are induced the intervention of diabetic mice to STZ

Cleaning level Kunming male mice, body weight 26-30 gram, adaptability is raised 5d, and behind the fasting 16h, tail vein is got blood and is measured fasting plasma glucose with blood glucose meter, selects for use blood glucose value to enter test less than the mouse of 6mol/L.Divide 5 groups at random by body weight, every group of 10 mouse, behind the fasting 16h, the following five groups of medicines of abdominal injection:

Group A: citrate buffer solution (Buffer)+physiological saline (0.9 quality %NaCl);

Group B:50mg/kg streptozotocin (STZ)+physiological saline (0.9 quality %NaCl); Group C:50mg/kg STZ+500 μ g/kg PACAP38;

The linear pentapeptide of group D:50mg/kg STZ+500 μ g/kg HSDGI (the linear pentapeptide of HSDGI is the His-Ser-Asp-Gly-Ile linear peptides);

Group E:50mg/kg STZ+500 μ g/kg CHC ring seven peptide.

Injection is 5 days continuously, measures the fasting plasma glucose of each group after 7 days.

The result is as shown in Figure 6: compare with the blank group of injection Buffer+ physiological saline, STZ+ physiological saline group blood sugar significantly raises (P＜0.01), illustrate that STZ induces diabetes model successfully; The blood sugar of STZ+PACAP38 group rises and receives slight inhibition, but does not have statistical significance; STZ+CHC ring seven peptide and STZ+HSDGI group all effectively (P＜0.01) suppressed the rising of blood sugar, show that CHC ring seven peptide and HSDGI linear peptides all can resist the damage of STZ to islet cells, and the CHC ring seven peptide is more better than the effect of HSDGI linear peptides.

Embodiment 5 ring seven peptides (CHC) reverse the effect of PACAP (6-38) antagonism nerve growth

Cultivate PC12 cell (the PC-12 cell is a neurocyte strain commonly used, and is commercially available), nutrient solution is the DMEM that contains 5% new-born calf serum and 10% horse serum, and putting 37 ℃, volume fraction is 0.05 CO ₂Cultivate in the incubator.

With PC 12 cell inoculations of 1 * 107/L in scribbling the 96 hole plastic culture plates of poly-l-lysine poly-L-lysine, every hole 100 μ l, treat cell attachment after, cell is divided into 6 groups at random:

A: blank;

B：PACAP38；

C、PACAP(6-38)；

D、CHC+PACAP(6-38)；

E、PACAP(6-38)+PACAP38；

F、CHC+PACAP(6-38)+PACAP38。

Cultivate 72h, observation of cell enation situation.Regulation cell process length is the enation positive cell more than or equal to 1 times of person of cell space diameter.150～200 cells of randomized number, the per-cent of calculating enation positive cell.Every group of data are from twice experiment.

The result as shown in Figure 7, PACAP (6-38) itself is to the not obviously effect of vitro culture neurocyte, but CHC+PACAP (6-38) effectively promote the PC12 enation ( ^*P＜0.01), play with PACAP38 similarly acting on, prompting CHC can effectively compensate the function of PACAP (6-38).

And PACAP (6-38) itself suppresses the short spinous process role (△ p＜0.01) of PACAP38, but after adding CHC, restraining effect is converted into promoter action, CHC+PACAP (6-38)+PACAP38 group than the nervous process per-cent of PACAP38 group higher ( ^*P＜0.01).

Above presentation of results CHC ring seven peptide has effective compensation and reverses the function of PACAP (6-38) effect.

Claims

1, a kind of small peptide, its aminoacid sequence are Histidine-Serine-aspartic acid-glycine-Isoleucine.

2, a kind of ring seven peptide that contains the described small peptide of claim 1, its structural formula is ring-(halfcystine-Histidine-Serine-aspartic acid-glycine-Isoleucine-halfcystine).

3, according to the described ring seven peptide of claim 2, the cyclisation mode that it is characterized in that this ring seven peptide is to form disulfide linkage between the 1st halfcystine and the 7th halfcystine, thereby becomes a ring texture.

4, a kind of method for preparing the described ring seven peptide of claim 2 is characterized in that this method is a solid-phase synthesis.

5, the application of the described small peptide of claim 1 in diagnosis or treatment and Pituitary adenylate cyclase-activating polypeptide., vasoactive intestinal peptide relative disease.

6, the application of the described ring seven peptide of claim 2 in diagnosis or treatment and Pituitary adenylate cyclase-activating polypeptide., vasoactive intestinal peptide relative disease.

7; according to claim 5 or 6 described each application, it is characterized in that described disease is diabetes; fat; obesity-related disease; metabolic disturbance; the metabolism syndromes of being correlated with; brain injury; cerebral ischemia; prevent nerve cell death; injured nerve cytoprotective and regeneration; anti-inflammatory; impotence; hyposexuality; infertile; Female sexual dysfunction; neurodynia; neuropathy; dizzy; anxiety disorder; psychosis; memory impairment; dull-witted; Cog Disorg; central nervous system disease; migraine; nerve is shunk; myocardial ischemia; myocardial infarction; myocardial fibrosis; arteriosclerosis; the flesh disease of shrinking; stomach ulcer; hypertension; interior toxicogenic shock; thrombosis; retinopathy; cardiovascular disorder; renal failure; heart failure; apoptosis of tumor cells or tumor cell proliferation.