CN1286264A - Action of proinsulin associated peptide on dopamine transporter and its application in treating correlative diseases - Google Patents

Abstract

The invention provides a proinsulin-related peptide with the function of regulating the activity of dopamine transporter and a preparation method thereof. Different proinsulin-related peptides have different regulation modes for dopamine transporter, so that bidirectional regulation of dopamine action can be achieved, so it can be used in the preparation of drugs for treating neurological and psychiatric diseases as well as peripheral diseases.

Description

The effect of proinsulin-related peptide on dopamine transporter and the treatment of related diseases

The present invention relates to the effect of polypeptide on dopamine transporter and the treatment of related diseases, more specifically proinsulin-related peptide and its preparation method, and its application in the preparation of medicines for treating neurological and psychiatric diseases and peripheral diseases.

The central dopamine transporter is located in the presynaptic membrane of dopaminergic neurons, and its main function is to regulate the concentration and distribution of dopamine in the synaptic cleft, affect the time course and intensity of synaptic signal transmission, and maintain the normal physiological functions of the dopaminergic nervous system ( Giros B et al. Nature 379:606-612). The pathology and etiology of many neurological and psychiatric diseases and cocaine addiction (Ritz M C et al.Science237:1219-1223) are related to the role of this protein, and many clinically used drugs are also based on this protein Targets, such as certain tricyclic antidepressants, certain anti-Parkinson's disease drugs (Boyson S J et al. Ann. Neurol. 30: 330-331), etc.

Insulin is a protein hormone naturally secreted in the body. Preproinsulin is synthesized by pancreatic beta cells and is quickly decomposed by enzymes. The proinsulin polypeptide is cut off to generate proinsulin, which is further enzymatically decomposed into insulin and C-peptide in the Golgi body. The molecule is secreted into the blood. Insulin is a specific drug for the treatment of diabetes (Chi Zhisheng, editor-in-chief Diabetology, 1982). There have been extensive and in-depth studies on the biological effects of insulin on traditional target organs such as liver and adipose tissue. Insulin also exists in the central nervous system. In 1978, Havrankova and his collaborators (Havrankova J et al. Proc Natl Acad SciUSA, 1978; 75:5737) first reported the presence of insulin in rat brain extracts. It was found that the average insulin concentration in the golden brain was 25 times higher than that in plasma, and the insulin content in different regions of the brain was significantly different. They also proved that insulin receptors widely exist in various regions of the central nervous system of rats. In addition, the existence of insulin mRNA in the brain also provides a biochemical basis for the viewpoint of insulin synthesis in situ (Boyd Jr F T et al.J Biol Chem, 1985, 260:15880). Studies over the years have indeed shown that insulin is an important regulator of the nervous system. Insulin can regulate the use of glucose in the brain, promote the development of the nervous system (Clarke D W et al. Am J Physiol, 1985; 249: C484), and regulate the neurotransmission activity of brain cells (Wolinsky E J.J Neurosci.1985; 5: 1675 ; Rhoads D E. Biochem Biophys Res Commun, 1984; 119: 1198), affecting animal feeding and body weight (Stein L J et al. Int J Obesity, 1985; 9: A31; Woods S C et al. Am J Clin Nutr , 1985;42:1063). Intraventricular injection of insulin can increase the mRNA level of dopamine transporter in the substantia nigra of rats (Figlewica DP et al. et al. Neuroendocrinology 1998 68: 11). In recent years, it has been reported that C-peptide combined with insulin can prevent nervous system dysfunction in patients with type 1 diabetes (Y. Ido, A. Vindingni et al: 1997 Prevention of vascular and dysfurction indiabetic rats by C-peptide. Science 277: 563-566). However, there are no previous reports on the detailed functions of proinsulin-related polypeptides and dopamine transporters and the treatment of related diseases.

The object of the present invention is to provide a class of proinsulin-related peptides with the function of regulating the activity of dopamine transporter; at the same time, it provides a preparation method of the peptides and its application in drugs for treating neurological and psychiatric diseases and peripheral diseases.

The "proinsulin-related peptide" in the present invention refers to a partial polypeptide sequence derived from proinsulin, its analogues and pharmaceutically acceptable salts. Unless otherwise specified, amino acids refer to the following 20 natural amino acids (the three-letter characters are: Gly, Ala, Asp, Glu, Asn, Gln, Ser, Thr, Leu, Ile, Lys, Arg, Phe, Tyr, Trp, Pro, Cys, Met, His, Val), including D-type and L-type; or unnatural amino acids, methylated amino acids, allo amino acids.

The present invention utilizes the established dopamine transporter cell model and the mouse striatum synaptosome preparation to identify the fragment of proinsulin that has an effect on the dopamine transporter. These fragments regulate the dopamine transporter in different ways, and can achieve bidirectional regulation of dopamine, and then can be clinically developed into drugs for the treatment of certain neurological and psychiatric diseases, such as Parkinson's disease, depression, anxiety wait.

Among the proinsulin-related peptides of the present invention, one type is a peptide derived from the C-terminus of insulin B chain, having the following sequence or partial sequence, and a pharmaceutically acceptable salt thereof:

X ₁ -X ₂ -Gly-Phe-Phe-Tyr-Thr-Pro-Lys-X ₃ -X ₄ (SEQ ID NO: 1 series)

Wherein _X1 is acetyl group, acetylated amino acid or deamino amino acid; _X2 and _X3 can be a single amino acid, or multiple amino acids or peptides; _X4 is amino, carboxyl or hydroxyl; the amino acid sequence of such peptides Contains at least the core structure: Tyr-Thr-Pro-Lys-X, where X is a single amino acid residue.

These proinsulin-related peptides have an inhibitory effect on the activity of the dopamine transporter. Among them, the peptide with strong inhibitory effect is: Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala

There is also a class of proinsulin-related peptides, which are polypeptides or pharmaceutically acceptable salts thereof after removing 2 to 11 amino acids at the C-terminal of the B chain, and its amino acid sequence (SEQ ID NO: 2 series): wherein the A chain: X ₁ - A(1-21)-X ₅ B chain: X ₆ -B(1-19)-X ₉

A stands for insulin A chain

X₂为Glu或Asp.X₃为Thr或Ala.X₄为Ile或Val.X₇为Asn或Lys.X₈为Ser或Pro.以下列出了胰岛素原C肽氨基酸序列(其中X_0x列出了人或鼠的氨基酸变化)：X₀₀-Glu-Ala-Glu-Asp-X₀₁-Gln-Val-X₀₂-Gln-X₀₃-Glu-Leu-Gly-Gly-Gly-Pro-Gly-Ala-Gly-X₀₄-Leu-Gln-X₀₅-Leu-Ala-Leu-Glu-X₀₆-X₀₇-X₀₈-Gln-Lys-Arg-X₀₀为乙酰基、乙酰化氨基酸或去氨基的氨基酸。X₀₁为Leu或Pro.X₀₂为Gly或Ala.X₀₃为Val或Leu.X₀₄为Ser或Asp.X₀₅为Pro或Thr．X₀₆为Gly或Val.X₀₇为Ser或Ala.X₀₈为Leu或Arg.其优选方案是B链C端去八肽人胰岛素(DOI)，即X₉是Gly-Glu-Arg。B stands for insulin B chain, the number of amino acids from N-terminal to C-terminal in brackets, A ₆ and A ₁₁ have intramolecular disulfide bonds of A chain, A ₇ and B ₇ , A ₂₀ and B ₁₉ have two pairs of A and B chain molecules Between disulfide bonds, X ₁ is acetyl, acetylated amino acid, deamino amino acid or insulin C peptide and peptide derived from C peptide; X ₅ is amino, carboxyl or hydroxyl X ₆ is acetyl, acetylated amino acid or deamino The amino acid _X9 of the amino group is any one of the following sequences from the N-terminal to 9 amino acid sequences: N-terminal Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-X ₀₉ X ₀₉ is amino, carboxyl or hydroxyl. Because of the different sources of species, amino acids will change, and the detailed sequence and amino acid changes of human or mouse are further listed below:

X ₂ is Glu or Asp.X ₃ is Thr or Ala.X ₄ is Ile or Val.X ₇ is Asn or Lys.X ₈ is Ser or Pro. The proinsulin C peptide amino acid sequence is listed below (wherein X _0x column Human or mouse amino acid changes): X ₀₀ -Glu-Ala-Glu-Asp-X ₀₁ -Gln-Val-X ₀₂ -Gln-X ₀₃ -Glu-Leu-Gly-Gly-Gly-Pro-Gly- Ala-Gly-X ₀₄ -Leu-Gln-X ₀₅ -Leu-Ala-Leu-Glu-X ₀₆ -X ₀₇ -X ₀₈ -Gln-Lys-Arg-X ₀₀ is acetyl, acetylated amino acid or deamino amino acid. X ₀₁ is Leu or Pro. X ₀₂ is Gly or Ala. X ₀₃ is Val or Leu. X ₀₄ is Ser or Asp. X ₀₅ is Pro or Thr. X ₀₆ is Gly or Val. X ₀₇ is Ser or Ala. X ₀₈ is Leu or Arg. The preferred solution is de-octapeptide human insulin (DOI) at the C-terminal of the B chain, that is, X ₉ is Gly-Glu-Arg.

The proinsulin-related peptides can promote the activity of dopamine transporter.

The present invention also provides a preparation method of proinsulin-related peptide, which is prepared by solid-phase or liquid-phase peptide chemical synthesis technology, or by genetic engineering method, or by enzymatic cleavage processing method. chemical synthesis

Conventional peptide chemical synthesis techniques can be used to synthesize the proinsulin-related peptides of the present invention by solid-phase or liquid-phase methods, but solid-phase synthesis is preferred (for example, see Birr, C., Aspect of the Merrifield Peptide Synthesis, Springer- Verlag, Heidelberg, 1978; Stewart et al., Solid Phase Peptide Synthesis, 2nd.ed., Pierce Chem.co., Rockford, IL, 1984; Barany, G. And Merrifield R.B. in The Peptides, Vol.2; Gross, E. & Meienhoffer J., eds., Academic Press, New York, pp3-284, 1979). Briefly, according to the designed and given amino acid sequence, the C-terminal amino acid residue of the peptide chain protected by an appropriate protecting group is connected to the solid phase support by using an appropriate activator and condensing agent. Depending on the amino acids to be linked, various solid phase supports for peptide synthesis can be used, for example including but not limited to polyethanol, divinylbenzene cross-linked polystyrene, and polyacrylamide resins. Resin selection

The appropriate resin carrier and synthesis system can be selected according to the sequence characteristics of the target peptide. For example, acid-sensitive resins can be used in the Fmoc system. For this purpose, preferred solid supports include PEG-PS-resins, divinylbenzene cross-linked polypropylene resins, different functional groups of such resins include chloromethyl resins, methylol resins, para-acetamidomethyl resins, Resin, 4-methylbenzhydrylamine (MBHA) resin, 4-(2',-4'-dimethoxyphenylaminomethyl)-phenoxymethyl resin, 2,4-dimethoxy Base benzylamine resin and (2,4-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyacetamido-norleucyl-MBHA resin (ie Rink Amide MBHA resin). A particularly preferred protecting group for α-amino acids according to the invention is the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group. Protected Amino Acid Selection

When using the Fmoc system for solid-phase synthesis, the following protected amino acid residues can be appropriately selected: Fmoc-Cys (Trt), Fmoc-Asn (Trt), Fmoc-Ser (But), Fmoc-Leu, Fmoc-Thr ( But), Fmoc-Val, Fmoc-Gly, Fmoc-Lys(Boc), Fmoc-Gln(Trt), Fmoc-Glu(Obut), Fmoc-His(Trit), Fmoc-Tyr(But), Fmoc-Arg( Pmc) or Fmoc-Arg(Pbf) and Fmoc-Pro. When using the Boc system for solid-phase synthesis, the following protected amino acid residues can be appropriately selected: Boc-Cys(4-Mzl), Boc-Lys(CLZ), Boc-His(Bom), Boc-Asp(OCHex) , Boc-Thr(Bzl), Boc-Ser(Bzl), Boc-Pro, Boc-Trp(CHO). Here, Fmoc or Boc is used to protect the α-amino groups of various amino acids, and the groups in brackets are used for Protect the side chains of amino acids. Coupling and Activation

The individual amino acid residues can be coupled using various coupling agents and coupling methods known in the field of peptide chemistry, for example dicyclohexylcarbodiimide (DCC) can be used for direct coupling, or via active esters such as pentafluoro Phenyl ester, or use Fmoc-amino acid-carboxylic acid anhydride. Hydroxybenzotriazole (HOBt) or 7-azahydroxybenzotriazole (HOAt) or 2-(1H-benzotriazol-1-yl), 1,1,3,3-tetra Urea hexafluorophosphate (HBTU) activates amino acids. Synthesis and Purification

The synthesis of the above-mentioned proinsulin-related peptides can be accomplished manually, or by using an automatic peptide synthesizer, such as an ABI 433A or PE Pioneer automatic peptide synthesizer produced by Applied Biosystems. In the Fmoc synthesis system, treat with 20% hexahydropyridine/dimethylformamide (DMF) at room temperature for 20 minutes to remove the Fmoc protecting group, and extend from the C-terminal to the N-terminal one by one according to the given amino acid sequence. After the synthesis is complete, the synthesized proinsulin-related peptide is cleaved from the resin with trifluoroacetic acid containing detergent and the protecting group is removed. The crude peptide can be obtained by filtering out the resin and then precipitating with ether. After lyophilization of the resulting product solution, the desired peptide was purified by gel filtration and reverse phase high pressure liquid chromatography. In the Boc synthesis system, the protecting group Boc was removed with TFA/dichloromethane (DCM) and neutralized with diisopropylethylamine (DIEA)/dichloromethane in a cycle of deprotection, neutralization, and coupling. After the peptide chain condensation is completed, treat with hydrogen fluoride (HF) containing p-cresol (5-10%) at 0° C. for 1-2 hours to cut the peptide chain from the resin and remove the protecting group at the same time. The peptide is extracted with 50-80% acetic acid (containing a small amount of mercaptoethanol), and the solution is freeze-dried, further separated and purified by molecular sieve Sephadex G10 or Tsk-40f, and then purified by high-pressure liquid phase to obtain the desired peptide.

And the proinsulin-related peptide prepared by the above-mentioned technique or recombinant DNA technique can be prepared into a pharmaceutically acceptable salt thereof by a known method. For example, suitable salts can be obtained by treating these peptides with appropriate acids and bases according to methods well known to those skilled in the art.

The present invention observes the effect of the proinsulin-related peptide on the dopamine transporter through the following experiments:

1. Proinsulin-related peptides act on the dopamine transporter permanently expressed on the membrane of Chinese hamster ovary cells (CHO), thereby promoting or inhibiting the uptake of dopamine by cells.

2. Proinsulin-related peptide acts on the dopamine transporter on striatal synaptosomes, thereby promoting or inhibiting the uptake of dopamine by the dopamine transporter.

3. Proinsulin-related peptides were microinjected into the substantia nigra of rats on one side, and the function of substantia nigra was affected by changing the function of dopamine transporter, so as to observe the influence on living rats.

It is confirmed that the proinsulin-related peptides of SEQ ID NO: 1 series have obvious inhibitory effect on the activity of dopamine transporter; the proinsulin-related peptides of SEQ ID NO: 2 series have obvious promotion effect on the activity of dopamine transporter. The action property of the mixture of 9-peptide and DOI in equimolar ratio is to inhibit the uptake of dopamine by dopamine transporter. In addition, the trypsin cleavage product of human insulin acts on the dopamine transporter permanently expressed on the membrane of Chinese hamster ovary cells (CHO) to promote the uptake of dopamine by the dopamine transporter (Figure 6), indicating that natural insulin degradation The possible effect of the drug on the dopamine transporter in vivo has confirmed that the partial polypeptide sequence derived from proinsulin has a bidirectional regulatory effect on the dopamine transporter.

Based on the above, the proinsulin-related peptide of the present invention can be applied to drugs for treating neurological and psychiatric diseases and peripheral diseases.

The proinsulin-related peptide of the present invention can be used to prepare a pharmaceutical composition for treating neurological and psychiatric diseases and peripheral diseases, which contains an effective amount of the proinsulin-related peptide and a pharmaceutically acceptable carrier.

Fig. 1 Determination of the molecular weight of the C-terminal 9-peptide of insulin B chain by electrospray mass spectrometry.

Theoretical molecular weight: 1086.26 Da; measured molecular weight: 1086.0 Da

Fig. 2 Determination of molecular weight of DOI by electrospray mass spectrometry.

Theoretical molecular weight: 4865.46 Da; measured molecular weight: 4865.0 Da

Fig. 3 The high-expression permanent expression line of dopamine transporter screened out.

D8 is a high expression dopamine transporter permanent expression line.

*: P<0.01

P value, Figure 4-Figure 6 are compared with D8

Fig. 4 Effect of different concentrations of 9-peptide on dopamine transporter on D8 cells.

1. CHO control

2. D8 control

3. 30ug/ml 9 peptides

4. 20ug/ml 9 peptides

5. 10ug/ml 9-peptide

6. 7.5ug/ml 9-peptide

7.5ug/ml 9-peptide

8. 2.5ug/ml 9-peptide

9. 1ug/ml 9-peptide

10. 0.5ug/ml 9-peptide

11. 0.25ug/ml 9-peptide

*: P<0.01

Figure 5 Effects of different concentrations of DOI on dopamine transporter on D8 cells.

A: 0.1ug/ml DOI

B: 0.2ug/ml DOI

C: 0.4ug/ml DOI

D: 1.0ug/ml DOI

E: 2.0ug/ml DOI

F: 4.0u/ml DOI

*: P<0.01

Figure 6 Equimolar ratio of 9-peptide and DOI mixture and trypsin digestion product of human insulin

Effects on the dopamine transporter on D8 cells.

A: CHO control

B: D8 control

C: 10ug/ml 9-peptide

D: 48ug/ml DOI

E: C+D

F: trypsin digestion product of 100ug/ml human insulin

G: trypsin digestion product of 20ug/ml human insulin

H: enzymatic digestion buffer

*: P<0.01

Figure 7. Effect of the 9-peptide on the dopamine transporter in striatal synaptosomes.

A: Transport of dopamine in striatal synaptosomes without 9-peptide.

B: Effect of 1 ug/ml of 9-peptide on dopamine transport.

C: Effect of 10 ug/ml of 9-peptide on dopamine transport.

All data were subtracted for negative controls (i.e., nonspecific adsorption of dopamine to striatal synaptosomes in a Li ⁺ environment).

*: P<0.01

P value, Figure 8-Figure 9 are compared with HBS buffer

Fig. 8 The effect of different concentrations of nine peptides injected into the right substantia nigra in vivo on the maximum rate of left turning in rats.

A: 1ul 450mM cocaine

B: 1ul 20ug/ul 9 peptides

C: 1ul 10ug/ul 9 peptides

D: 1ul 5ug/ul 9 peptide

E: 1ul 2.5ug/ul 9 peptides

F: 1ul HBS buffer

The Y-axis in the figure represents the maximum number of turns per minute of the rat's left turn (each point in the figure is the average value of three rats).

*: P<0.001

Figure 9 The effect of different concentrations of nine peptides injected into the right substantia nigra in vivo on the duration of left turn in rats.

A: 1ul 450mM cocaine

B: 1ul 20ug/ul 9 peptides

C: 1ul 10ug/ul 9 peptides

D: 1ul 5ug/ul 9 peptide

E: 1ul 2.5ug/ul 9 peptides

F: 1ul HBS buffer

*: P<0.01

The present invention will be further elaborated below in conjunction with specific examples, but the content of the present invention is not limited. Example 1 Synthesis of proinsulin-related peptides using the Fmoc system

Take the C-terminal 9-peptide of insulin B chain as an example: NH ₂ -Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys-Ala-OH

The starting Fmoc-Ala-PAC-PEG-HL resin (1 g, 0.45 mmol) was filled in a 30 ml vial and treated with DCM for 30 minutes. Condensate each protected amino acid one by one from the C-terminus to the N-terminus as shown in the table below. After each step of the reaction is completed, the reagents are removed by filtration under negative pressure and separated from the peptide resin. Wherein, after the condensation reaction between the first protected amino acid Fmoc-Ala at the C-terminal and the resin is completed, the condensation of the remaining amino acids is carried out as shown in Tables 1-13.

Each amino acid condensation reaction was carried out for an average of 2 hours. Before entering the next amino acid condensation cycle, use the ninhydrin method to measure the free amino group to determine whether the condensation is complete. According to the above reaction steps, the peptide chain is extended from the C-terminal amino acid to the N-terminal one by one to obtain the target peptide chain resin, and the resin is removed from the reactor and then vacuum-dried.

Dry peptide resin (0.5 g) and p-cresol (0.5 ml) were placed in the reactor and pre-cooled in an ice bath for 30 minutes. Add 0.5ml of water, 9ml of TFA and stir the reaction at room temperature for 1 hour. The reaction solution was filtered off, the residual resin was washed three times with a small amount of TFA, and then the filtrates were combined. DCM and TFA were removed by evaporation under vacuum. When the liquid is basically drained and becomes viscous, add pre-cooled ethyl acetate, shake quickly and gradually precipitate the crude peptide in solid form and centrifuge to precipitate it. The crude peptide was purified by TSK-SW G-300 column molecular sieve chromatography and C ₈ column reversed-phase high-performance liquid phase to obtain the target peptide, and its molecular weight was determined to be 1086 by mass spectrometry, which was consistent with the theoretical value of 1086.4 (Figure 1), and the complete sequence of amino acids was determined. The arrangement is accurate, indicating that the obtained polypeptide has a precise structure. This method can also be used for the synthesis of other proinsulin-related peptides. Step Reagent Reagent Consumption (ml) Repeat Times Mixing Time (Min) 1 DMF 20 3 22 Hexahydropyridine/DMF 20 53 Hexahydropyridine/DMF 20 154 DMF 20 3 25 DCM 20 26 DMF 20 3 2Fmoc-AA, 1.8mmol7 HOBT /DCC 15 120 dissolved in DMF8 DCM 20 3 29 Ethanol 20 3 210 DCM 20 3 211 DMF 20 3 2Fmoc-AA, 1.5mmol12 HOBT/DCC 15 120 dissolved in DMF13 Repeat 8-11 steps 14 Repeat 1-13 to complete the C-terminal amino acid Extending to the N-terminal one by one Example 2 Preparation of B-chain C-terminal desoctopeptide insulin (DOI)

Dissolve de-Zn ⁺ porcine insulin or human proinsulin expressed by genetic engineering in 0.02M borax, 0.001M calcium chloride solution, the insulin concentration is 5 mg per ml, add trypsin (Trypsin) treated with TPCK, between the enzyme and the substrate The weight ratio is 1:50, and the reaction is carried out at 37° C. for 3-8 hours. Adjust the pH to 4.8, place overnight at 0°C, and centrifuge. Then, DEAE-Sepharose Cl-6B ion-exchange chromatography and Nacl salt gradient elution were used to collect the main peak DOI, which was desalted with Sephadex G-25 and freeze-dried to obtain pure DOI. The molecular weight was determined by electrospray mass spectrometry, and the measured value was 4865.0, which was consistent with the theoretical value of 4865.46 (Figure 2). Example 3 Construction of dopamine transporter cell model

Chinese hamster ovary tumor (CHO) cells were cultured in 1640 medium containing 10% calf serum in a 5% CO ₂ incubator at 37°C. Cells in logarithmic phase of growth were taken for transfection. Taking the dopamine transporter as an example, import cells by electroporation: wash the cells once with PBS buffer, digest with 0.125% trypsin, wash twice with PBS buffer, and then blow the cells with PBS to suspend the cells Then transfer to a sterile centrifuge tube and centrifuge (1000rpm×8min). Discard the supernatant, wash the precipitated cells twice with electrotransfer solution (272mM sucrose, 7mM sodium phosphate, 1mM magnesium chloride, pH7.4) in ice bath, then add ice bath cold electrotransfer solution to suspend the cells, count about ml 10 ⁷ pcs. Divide into two 1ml electric shock cups, 0.8ml/each. One group is a negative control, add about 10ul of blank vector pCDNA3, and the final concentration of pCDNA3 is 2-10ug/ml. The other group is the experimental group, about 10ul of DAT/pCDNA3 is added, and the final concentration of DAT/pCDNA3 is 2-10ug/ml. Ice bath for 15 minutes, then put the electric shock cup into the electric shock instrument, set as: voltage 350v, capacitance 25uFD, discharge pulse once, about 10ms. The electric shock cup was put back into the ice bath for 10 minutes. Finally, the solution in each electric shock cup was divided into three parts, inoculated into three 25ml culture bottles, added 3ml of 1640 culture solution, and cultured in a 37°C, 5% _CO2 incubator. After 48 hours, culture with 1640 medium containing 200-600ug/ml G418. After 10 days, all the cells in the control group died, while many cell clones formed in the experimental group. Pick up the cell clones in a 96-well plate with a pipe tip that is cut flat at an angle, and place each cell clone in one well. The medium used thereafter contains 200-600ug/ml G418 1640 medium, and after culturing for one week The cells covered the bottom of the well, the culture medium was sucked off, digested with trypsin as above, and the cells in each well were inoculated into corresponding wells of two 96-well plates. After the cells covered the bottom of the well, one of the plates was used for isotope flow measurement, washed once with PBS, sucked off the PBS solution, added 90ul HBS (10mM Hepes, 100mM NaCl, pH8.0) to each well, and warmed at 25°C-37°C. After incubation for 10 minutes, 10ul of HBS reaction solution (containing 10uCi/ml ³ H-DA, 1mM vitamin C, 1mM pargyline) was added to each well. Incubate at 25°C-37°C for 20 minutes, wash three times with ice-bathed PBS solution, lyse with 100ul 2N NaOH or 1% SDS solution for 30 minutes, pipette the lysate from each well and add it to 1.6ml scintillation solution (PPO 3.6g , POPOP 0.36g, xylene 600ml, Triton X-100 300ml), put into liquid scintillation counter to detect the content of isotope, measure the activity of dopamine transporter with this. The cells corresponding to the wells with high transport function in the other plate were expanded and cultured step by step, and the isotope flux was measured at each level, and the cell line with higher transport activity was selected for culture, and finally the cell clone with the highest transport activity was selected for preservation (see below Called D8, Figure 3). Example 4 The effect of insulin B-chain C-terminal 9-peptide (hereinafter referred to as 9-peptide) on the dopamine transporter permanently expressed on the membrane of Chinese hamster ovary cells (CHO)

Inoculate Chinese hamster ovary cells (CHO) permanently expressing high-transport activity dopamine transporter in 24 or 48 or 96-well plates until the cells cover the bottom of the well. Wash once with PBS, absorb PBS (8g NaCl, 0.2g KCl, 3.6g Na ₂ HPO ₄ 12H ₂ O, 0.24g KH ₂ PO ₄ , dissolve in 1000ml water, adjust pH to 7.4) solution, add 90 μg liter of HBS (10mM Hepes, 100mM NaCl, pH8.0), incubate at 25°C-37°C for 10 minutes, add 10 μl of HBS reaction solution (containing 10 μCi/ml 3H-DA, 1mM vitamin C, 1mM pargyline ). Incubate at 25°C-37°C for 20 minutes, wash three times with ice-bathed PBS solution, lyse with 2N NaOH or 1% SDS solution for 30 minutes, pipette the lysate from each well and add it to 1600 microliters of scintillation fluid (PPO 3.6g , POPOP 0.36g, xylene 600ml, Triton X-100 300ml), put into liquid scintillation counter to detect the content of isotope, measure the activity of dopamine transporter with this. When studying the effect of 9-peptide on the dopamine transporter, it is only necessary to add a certain volume of HBS under the condition of maintaining a reaction system of 100 microliters, and supplement the corresponding volume of 9-peptide at different concentrations, and the other is the same. Judging from the change in the activity of the dopamine transporter, the nature of the action of the 9-peptide is to inhibit the uptake of dopamine by the dopamine transporter ( FIG. 4 ). Example 5 Effect of DOI on dopamine transporter permanently expressed on Chinese hamster ovary cell (CHO) membrane

Refer to the operation steps of Example 4, replace the 9-peptide with DOI, and the others are the same. Judging from the change in the activity of the dopamine transporter, the action property of DOI is to promote the uptake of dopamine by the dopamine transporter ( FIG. 5 ). Example 6 The effect of the mixture of 9 peptides and DOI in equimolar ratio on the dopamine transporter permanently expressed on the Chinese hamster ovary cell (CHO) membrane

Refer to the operation steps of Example 4, replace the 9-peptide with the mixture of 9-peptide and DOI, and the others are the same. Judging from the change in the activity of the dopamine transporter, the nature of the action of the mixture of equimolar ratio of 9-peptide and DOI is to inhibit the uptake of dopamine by the dopamine transporter ( FIG. 6 ). Example 7 The effect of the trypsin digestion product of human insulin on the dopamine transporter permanently expressed on the Chinese hamster ovary cell (CHO) membrane

See Example 2 for the enzymatic digestion conditions of human insulin. To study the effect of human insulin trypsin cleavage product on dopamine transporter, refer to the operation steps in Example 4. Judging from the change in the activity of the dopamine transporter, the action of the trypsin digested product of human insulin is to promote the uptake of dopamine by the dopamine transporter ( FIG. 6 ). Example 8 The effect of insulin B chain C-terminal 9-peptide on dopamine transporter on striatal synaptosomes

The mice were sacrificed by neck dislocation, the whole brain was exposed, the striatum was isolated, and the blood and other dirt were washed with isotonic sucrose. Weigh on a smooth small piece of paper weighed in advance, put it into a pre-cooled glass homogenizer, add 9 times the volume (1g tissue/9ml), that is, 1:9 (W/V) 0.32M sucrose/PBS solution Homogenize, up and down 10-12 times, all operations are carried out in an ice bath at 0-4°C. Transfer the homogenate into a pre-cooled centrifuge tube, centrifuge at 0°C, 1000×12min, _take the ^supernatant and centrifuge as above, and the precipitate is the crude synaptosome part _. , 1mM KH ₂ PO ₄ , 27mM NaHCO ₃ , 5.4mM Glucose, pH 7.4) solution suspension, pay attention to pipetting with a cut tip. Add 10 μl ³ H-DA (1 μCi/ml) and add 10 μl of different concentrations of 9-peptide, 70 μl Na ⁺ -KRH, add 10 μl of synaptosome suspension, and incubate at 37°C for 10 min. Negative control: use Li ⁺ -KRH (103mM LiCl, 1 mM CaCl ₂ , 1 mM MgCl ₂ , 1 mM KH ₂ PO ₄ , 27 mM NaHCO ₃ , 5.4 mM Glucose, pH 7.4) instead of Na ⁺ -KRH. The reaction was terminated by adding 1.2 ml Li ⁺ -KRH in an ice bath. Centrifuge at 4°C, 10000×10min, discard the supernatant, wash once with Li ⁺ -KRH, centrifuge as above, discard the supernatant, add 50μl 2N NaOH for 30min at room temperature. Add 1.2ml of scintillation fluid, shake well, put it into a scintillation cup, and measure CPM with a liquid scintillation counter to measure the activity of dopamine transporter on striatal synaptosomes. It shows that the 9-peptide inhibits the uptake of dopamine by the dopamine transporter on the striatal synaptosome ( FIG. 7 ). Example 9 In vivo unilateral substantia nigra injection of insulin B chain C-terminal 9-peptide effect on rats

Take SD rats, male, mature, 200g-300g. Weigh, intraperitoneally inject 2% tribromoethanol (8ml/kg), and after anesthesia, position according to the George Paxions brain atlas with a stereotaxic instrument. Before the operation, the rats were fixed on the stereotaxic apparatus with a head clip, and the corresponding parts of the skull including the bregma and bregma were exposed according to routine procedures such as skin incision. Adjust the head clamp so that the height difference between bregma and bregma is 1 mm (the error is 0.2 mm, bregma is higher than bregma), drill a hole on the surface of the skull corresponding to the position of right substantia nigra, and insert the needle of the microinjector (positioning: behind bregma) 5.2mm, 2.2mm on the right side, 7.8mm in depth). After the rats woke up, 1 microliter of different concentrations of nine peptides (dissolved in HBS solution) was injected uniformly within 1 minute, and the needle was retained for 10 minutes. Take out the injection needle slowly, suture the wound, and wipe it with 75% alcohol cotton ball for disinfection. Cocaine instead of 9-peptide was used as a positive control, and HBS solution without 9-peptide was used as a negative control. Observe the reaction of the rat and find that the rat turns to the left in a circle, calculate the speed of the circle (Figure 8) and the time to return to normal (Figure 9). It shows that the 9-peptide inhibits the uptake of dopamine by the dopamine transporter, thereby causing the accumulation of dopamine in the synaptic cleft. This inhibitory effect has a dose-effect relationship with the concentration of 9-peptide.

Claims (6)

1. The proinsulin-related peptide is characterized by a polypeptide derived from proinsulin and a pharmaceutically acceptable salt thereof. 2. The proinsulin-related peptide according to claim 1, characterized in that it is derived from the C-terminus of the B chain and has the following amino acid sequence or partial sequence: X ₁ -X ₂ -Gly-Phe-Phe-Tyr-Thr-Pro-Lys-X ₃ -X ₄ Wherein X ₁ is acetyl, acetylated amino acid or amino acid deamino; X ₂ and X ₃ can be a single amino acid, or multiple amino acids or peptides, X ₄ is amino, carboxyl or hydroxyl; unless specified amino acid, all Refers to the following: 20 kinds of natural amino acids (three letters: Gly, Ala, Asp, Glu, Asn, Gln, Ser, Thr, Leu, Ile, Lys, Arg, Phe, Tyr, Trp, Pro, Cys, Met, His ,Val), including D-type and L-type; or unnatural amino acid, methylated amino acid, allo amino acid. 3. The proinsulin-related peptide according to claim 2, characterized in that its amino acid sequence contains at least the core structure: Tyr-Thr-Pro-Lys-X Wherein X is a single amino acid residue, which is 20 kinds of natural amino acids, including D-type and L-type; or one of unnatural amino acids, methylated amino acids, and allo amino acids. 4. The proinsulin-related peptide according to claim 1, which is characterized in that it is a polypeptide after removing 2 to 11 amino acids at the C-terminal of the B chain or a pharmaceutically acceptable salt thereof, and the amino acid sequence: wherein, the A chain: X ₁ - A(1-21)-X ₅ B chain: X ₆ -B(1-19)-X ₉ A stands for insulin A chain B means insulin B chain, the number of amino acids from N-terminal to C-terminal in brackets, A ₆ and A ₁₁ have intramolecular disulfide bonds of A chain, A ₇ and B ₇ , A ₂₀ and B ₁₉ have two pairs of A and B chain molecules Between disulfide bonds, X ₁ is acetyl, acetylated amino acid, deamino amino acid or insulin C peptide and peptide derived from C peptide; X ₅ is amino, carboxyl or hydroxyl X ₆ is acetyl, acetylated amino acid or deamino The amino acid _X9 of the amino group is any one of the following sequences from the N-terminal to 9 amino acid sequences: N-terminal Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-X ₀₉ X ₀₉ is amino, carboxyl or hydroxyl. 5. The preparation method of proinsulin-related peptides according to claims 1 to 4, characterized in that said peptides are synthesized by solid-phase or liquid-phase peptide chemical synthesis techniques, or by genetic engineering methods, or by enzymatic cleavage processing methods made. 6. The proinsulin-related peptide according to claims 1 to 4 has the function of inhibiting or promoting dopamine transporter, and can be used in the preparation of drugs for treating neurological and psychiatric diseases and peripheral diseases.

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