CN101434649B

CN101434649B - Cyclohepatapeptide, preparation thereof and use

Info

Publication number: CN101434649B
Application number: CN2008102198844A
Authority: CN
Inventors: 余榕捷
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-12-12
Filing date: 2008-12-12
Publication date: 2012-04-25
Anticipated expiration: 2028-12-12
Also published as: CN101434649A; WO2010066125A1

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 ring seven peptide

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 cns, peripheral nervous system; And in non-nervous tissue, also extensively exist, like 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; The energy metabolism balance is regulated in participation digestion activity; 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 VIP (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 receives 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, and its resistance to enzymolysis ability force rate line style peptide is strong, so can consider improving 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 to deficiency to 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 processed 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 the relevant disease of diagnosis or treatment and PACAP, VIP of above-mentioned small peptide or ring seven peptide.

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

The present invention is through a large amount of discovering; Small peptide Histidine-Serine-aspartic acid-glycocoll-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 designs some amino acid or group (like halfcystine or methyl) through the two ends at this pentapeptide; Make that the peptide chain after generating is a cyclic peptide structures; Then not only can play and regulate APCAP, the active effect of VIP, 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-glycocoll-Isoleucine-halfcystine) [cyclo-(Cys-His-Ser-Asp-Gly-Ile-Cys)].

In polypeptide was synthetic, peptide bond of every generation all need pass through protective group, condensation, go 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 compound 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 the Fmoc method here 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 like straight chain C ys-His-Ser-Asp-Gly-Ile-Cys is exactly before shearing, and the sulfydryl that Fmoc is protected carries out the oxidation on the resin, thereby linear pentapeptide is transformed into the ring seven peptide structure; Oxygenant can be for any one oxygenant, like I ₂, H ₂O ₂, Hg ²⁺Salt, K ₃Fe (CN) ₆, thallium trifluoroacetate T ₁(tfa) ₃, chlorosilane-sulfone class oxygenant or DMSO 99.8MIN. 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 VIP 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 to diagnose and treatment and PACAP, disease that VIP is relevant; Because PACAP, VIP are very conservative in the organic evolution process; Has extensive and important biological function; So the disease that ring seven peptide of the present invention possibly treated covers neural system, endocrine system, digestion and energy metabolism, The cardiovascular circulation, reproductive system and immunity system; Like mellitus (comprising I type, II type), obesity, obesity-related disease, metabolic disturbance, metabolism relevant syndromes, 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, psychosis, memory impairment, dementia, Cog Disorg, central nervous system disease, migraine, nerve are shunk, myocardial ischemia, myocardial infarction/fibrosis, arteriosclerosis, flesh are shunk disease, 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; Amino acid or group are added in the two ends that are employed in the 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 in a reaction vessel, carry out all reactions, 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 treatment and PACAP, disease that VIP is relevant; In view of PACAP, VIP very conservative in the organic evolution process; And has an extensive and important biological function; Therefore the medical value of ring seven peptide height, and this ring seven peptide promptly can obtain through 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, * representes P＜0.01;

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

Wherein, A is a Buffer+ saline water, and B is a STZ+ saline water, and C is STZ+PACAP38, and D is STZ+HSDGI, and E is STZ+ ring seven peptide CHC, and △ representes P＜0.01 (B group vs.A group), and * representes 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; C is PACAP (6-38), and D is CHC+PACAP (6-38), and E is PACAP (6-38)+PACAP38; F is CHC+PACAP (6-38)+PACAP38; △ representes P＜0.01 (B group vs.E group), and * representes P＜0.01 (D group vs.C group), and * * representes 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 following:

(1) by conventional Fmoc solid-phase synthesis synthesizing linear seven peptides: 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 N DMF mixed solution, be made into 0.1mol/L I ₂Solution), mixing adds in the reaction vessel, nitrogen blowing reaction 6h, and oxidation-SH generates 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 reaction density is high, 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 like Fig. 1, shown in 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), provided by No.1 Military Medical Univ.'s Experimental Animal Center, body weight 25 ± 5g divides 2 groups at random by body weight; Every group 10, difference abdominal injection 500 μ g/kg CHC ring seven peptides and saline water (0.9 quality %NaCl), at 0min, 10min; 20min, 30min, 45min and 60min measure blood sugar; The result is as shown in Figure 3, and 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, with 1.8mmol glucose/kg+ saline water (Saline) as contrasting, in 0min; 15min, 30min, 45min and 60min measure blood sugar concentration; The blood sugar concentration graphic representation is 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, can find out that through Fig. 4 and Fig. 5 ring seven peptide CHC has significant promoter action to sugar tolerance, and 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, flexibility 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 get into 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, five groups of medicines below the abdominal injection:

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

Group B:50mg/kg streptozotocin (STZ)+saline water (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+ saline water, STZ+ saline water group blood sugar significantly raises (P＜0.01), explains that STZ induces the diabetes model success; 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 PC 12 cells (the PC-12 cell is a neurocyte strain commonly used, and is commercially available), nutrient solution is the DMEM that contains 5% NBCS and 10% horse serum, and putting 37 ℃, volume(tric)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 is 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 promotes PC 12 enations (* p＜0.01), plays with PACAP38 similarly acting on, and 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, and CHC+PACAP (6-38)+PACAP38 group is than the nervous process per-cent higher (* * p＜0.01) of PACAP38 group.

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

Claims

1. ring seven peptide; Its structural formula is ring-(halfcystine-Histidine-Serine-aspartic acid-glycocoll-Isoleucine-halfcystine); The cyclisation mode of said ring seven peptide is to form disulfide linkage between the 1st halfcystine and the 7th halfcystine, thereby becomes a ring texture.

2. a method for preparing the described ring seven peptide of claim 1 is characterized in that this method is a solid-phase synthesis.

3. the application of the described ring seven peptide of claim 1 in preparation treatment mellitus, memory impairment, dementia or neural medicine of shrinking.