阅读说明：本技术 一种具有nNOS-Capon解偶联活性的多肽及其应用 (Polypeptide with nNOS-Capon uncoupling activity and application thereof ) 是由 秦亚娟 厉廷有 冯玲玲 徐剑 郑礼平 于 2020-04-15 设计创作，主要内容包括：一种具有nNOS-Capon解偶联活性的多肽及其应用,其特征在于氨基酸序列结构通式为Che-Xaa<Sup>1</Sup>-Xaa<Sup>2</Sup>-Xaa<Sup>3</Sup>-Val,其中Che=N-Cyclohexylethyl,Xaa<Sup>1</Sup>=Ala或Gly,Xaa<Sup>2</Sup>=Glu、Glu(OMe)、Asp或Asp(OMe),Xaa<Sup>3</Sup>=修饰的或天然氨基酸。这类多肽具有nNOS-Capon解耦连活性,可作为神经保护剂用于缺血性脑卒中的治疗。提供了一种体外快速筛选nNOS-Capon解偶联活性的荧光偏振法(FP)。通过FP法筛选的多肽在大鼠脑缺血再灌注模型(MACO)有明显神经保护作用。(The polypeptide with nNOS-Capon uncoupling activity and the application thereof are characterized in that the general structural formula of the amino acid sequence is Che-Xaa 1 ‑Xaa 2 ‑Xaa 3 -Val, wherein Che = N-Cyclohexylethyl, Xaa 1 = Ala or Gly, Xaa 2 = Glu, Glu (OMe), Asp or Asp (OMe), Xaa 3 = modified or natural amino acids. This is achieved byThe polypeptide-like has nNOS-Capon decoupling activity, and can be used as a neuroprotective agent for treating ischemic stroke. Provides a fluorescence polarization method (FP) for rapidly screening the uncoupling activity of nNOS-Capon in vitro. The polypeptide screened by the FP method has obvious neuroprotective effect on a rat cerebral ischemia-reperfusion Model (MACO).)

1. The polypeptide with nNOS-Capon uncoupling activity is characterized in that the general structural formula of an amino acid sequence is

Che-Xaa¹-Xaa²-Xaa³-Val, wherein chen-Cyclohexylethyl, Xaa¹Ala or Gly, Xaa²＝Glu、

Glu (OMe), Asp or Asp (OMe), Xaa³Modified or natural amino acids.

2. The polypeptide having nNOS-Capon uncoupling activity according to claim 1, characterized by the structural formula of any of the following compounds:

Che-Ala-Glu-Ala-Val、Che-Ala-Glu(OMe)-Ala-Val、

Che-Ala-Glu-Trp-Val、Che-Ala-Glu(OMe)-Trp-Val、

Che-Ala-Asp-Trp-Val、Che-Ala-Asp(OMe)-Trp-Val、

Che-Ala-Glu-Phe-Val、Che-Ala-Glu(OMe)-Phe-Val、

Che-Ala-Asp-Ala-Val、Che-Ala-Asp(OMe)-Ala-Val、

Che-Ala-Glu-Ile-Val、Che-Ala-Glu(OMe)-Ile-Val、

Che-Ala-Asp-Ile-Val、Che-Ala-Asp(OMe)-Ile-Val、

Che-Gly-Asp-Ala-Val、Che-Gly-Asp(OMe)-Ala-Val、

Che-Gly-Asp-Pro-Val、Che-Gly-Asp(OMe)-Pro-Val、

Che-Gly-Asp-Leu-Val、Che-Gly-Asp(OMe)-Leu-Val、

Che-Gly-Asp-Phe-Val、Che-Gly-Asp(OMe)-Phe-Val、

Che-Gly-Asp-Trp-Val or Che-Gly-Asp (OMe) -Trp-Val.

3. Use of a polypeptide as claimed in claim 1 or claim 2, or a pharmaceutically acceptable salt thereof, in the manufacture of a neuroprotective medicament.

4. Use of the polypeptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for stroke, anxiety or depression.

5. A neuroprotective agent characterized by comprising the polypeptide of claim 1 or 2 or a pharmaceutically acceptable salt thereof as an active ingredient.

Technical Field

The invention belongs to the field of pharmacy, and particularly relates to a polypeptide with nNOS-Capon uncoupling activity and application thereof.

Background

Ischemic stroke is one of the most major disabling and lethal diseases in the world, seriously harms human health, and the development of therapeutic drugs thereof is one of the most important subjects of medicinal chemistry. At present, the main researches on cerebral apoplexy drugs are as follows: calcium channel antagonists, glutamate release inhibitors, GABA receptor agonists, nNOS inhibitors, radical scavengers, MMP-9 inhibitors, NMDAR antagonists, etc., but the therapeutic effect is not ideal. Many neuroprotective agents that are effective in animal models of stroke fail to achieve the desired therapeutic effect in human clinical trials or have to be terminated because of the side effects that are too great.

The toxic effects of Excitatory Amino Acids (EAAs) play an important role in the pathogenesis of ischemic stroke when it occurs. Research shows that the NMDAR-PSD95-nNOS signal channel mediates the toxic action of cerebral apoplexy excitatory glutamic acid. However, since NMDAR and nNOS mediate many important physiological functions, inhibition of their activities can produce many toxic side effects. For example, clinical studies have shown that NMDAR antagonists cannot be developed as therapeutic drugs due to their side effects. When excitotoxic stimulation is carried out, a stimulation signal is transmitted to nNOS through NMDAR-PSD95-nNOS, nNOS is coupled with Capon, and Capon can be further coupled with MKK3 to form a nNOS-Capon-MKK3 ternary complex. nNOS can activate MKK3, thereby activating the p38MAPK signaling pathway, leading to neuronal cell death. Research shows that the coupling of nNOS-Capon is inhibited to have the neuroprotective effect.

The nNOS PDZ domain contains 127 amino acid residues, and Capon is a natural ligand for nNOS. Capon binds between the α B helix and β B sheet of the nNOS PDZ domain via its carboxy-terminal tetrapeptide EIAV, a shallow, long groove containing a binding pocket consisting of the conserved sequence GLGF (Gly21, Leu 22, Gly23, Phe 24). The polypeptides Che-A/G-D/E-X-V and Che-A/G-D/E (OMe) -X-V designed according to the carboxyl terminal EIAV of Capon play a role in nNOS-Capon uncoupling by competitively binding with a GLGF region pocket. The nNOS-Capon uncoupling activity of the polypeptide is screened and tested by a fluorescence polarization method, the structure of the compound with good activity is optimized by esterification after the compound with good activity is screened, the drug forming property of the compound is improved, and the neuroprotective activity of the compound is tested by a MACO animal model.

Disclosure of Invention

The technical problem to be solved is as follows: the invention provides a polypeptide with nNOS-Capon uncoupling activity and application thereof, which can be used as a neuroprotective agent for treating ischemic stroke.

The technical scheme is as follows: the polypeptide with nNOS-Capon uncoupling activity has an amino acid sequence general formula of Che-Xaa¹-Xaa²-Xaa³-Val, wherein chen-Cyclohexylethyl, Xaa¹Ala or Gly, Xaa²Glu, Glu (OMe), Asp or Asp (OMe), Xaa³Modified or natural amino acids.

A compound of any one of the following structural formulae:

Che-Ala-Glu-Ala-Val(Che-AEAV)、Che-Ala-Glu(OMe)-Ala-Val(Che-AE(OMe)AV)、

Che-Ala-Glu-Trp-Val(Che-AEWV)、Che-Ala-Glu(OMe)-Trp-Val(Che-AE(OMe)WV)、

Che-Ala-Asp-Trp-Val(Che-ADWV)、Che-Ala-Asp(OMe)-Trp-Val(Che-AD(OMe)WV)、

Che-Ala-Glu-Phe-Val(Che-AEFV)、Che-Ala-Glu(OMe)-Phe-Val(Che-AE(OMe)FV)、

Che-Ala-Asp-Ala-Val(Che-ADAV)、Che-Ala-Asp(OMe)-Ala-Val(Che-AD(OMe)AV)、

Che-Ala-Glu-Ile-Val(Che-AEIV)、Che-Ala-Glu(OMe)-Ile-Val(Che-AE(OMe)IV)、

Che-Ala-Asp-Ile-Val(Che-ADIV)、Che-Ala-Asp(OMe)-Ile-Val(Che-AD(OMe)IV)、

Che-Gly-Asp-Ala-Val(Che-GDAV)、Che-Gly-Asp(OMe)-Ala-Val(Che-GD(OMe)AV)、

Che-Gly-Asp-Pro-Val(Che-GDPV)、Che-Gly-Asp(OMe)-Pro-Val(Che-GD(OMe)PV)、

Che-Gly-Asp-Leu-Val(Che-GDLV)、Che-Gly-Asp(OMe)-Leu-Val(Che-GD(OMe)LV)、

Che-Gly-Asp-Phe-Val(Che-GDFV)、Che-Gly-Asp(OMe)-Phe-Val(Che-GD(OMe)FV)、

Che-Gly-Asp-Trp-Val (Che-GDWV) or Che-Gly-Asp (OMe) -Trp-Val (Che-GD (OMe) WV).

Preferred nNOS-Capon uncoupling polypeptides have the following structure:

the amino acid of the invention adopts an amino acid fragment modification method combined with a traditional solid phase synthesis method, and the synthetic route is shown in figures 1 and 2.

The application of the polypeptide or the pharmaceutically acceptable salt thereof in preparing neuroprotective drugs.

The polypeptide or pharmaceutically acceptable salt thereof can be applied to preparation of medicines for treating apoplexy, anxiety or depression.

A neuroprotective medicine contains the above polypeptide or its pharmaceutically acceptable salt as effective component.

Has the advantages that: the polypeptide provided by the invention has an obvious neuroprotective effect on a rat cerebral ischemia reperfusion Model (MACO), wherein the area of the cerebral infarction of a TTC staining model group of a MACO model brain section is 24.6 percent, the area of the Che-AD (OMe) AV cerebral infarction is 4.5 percent, and the area of the cerebral infarction of the edaravone of a positive control group is 13.5 percent.

Drawings

FIG. 1 is a synthetic diagram of alkylated amino acids;

FIG. 2 is a synthetic diagram of an alkylated tetrapeptide;

FIG. 3: GST-nNOS_1-133Western blot identification. Expression, purification and concentration of protein, SDS-PAGE electrophoresis, subsequent Coomassie brilliant blue staining solution staining to identify GST-nNOS_1-133。

FIG. 4: 5-FAM-KV-14 and GST-nNOS_1-133Schematic of integrated ITC. Isothermal Titration Calorimetry (ITC) was used to fit fluorescent molecular probes 5-FAM-KV-14 to GST-nNOS_1-133The binding process of the protein is carried out,the combination is verified to be a typical single-site combination model, and the screening requirement of the fluorescence polarization method is met.

FIG. 5: concentration schematic of optimal fluorescent molecular probe. The FP value decreased with increasing concentration of 5-FAM-KV-14. When the concentration of 5-FAM-KV-14 is 50nM, the FP value detected at this time is 57.25mP, and the FP value tends to be stable with the increase of the probe concentration, so that the concentration of 5-FAM-KV-14 finally selected is 50 nM.

FIG. 6: optimum GST-nNOS_1-133Schematic determination of protein concentration. With GST-nNOS_1-133The FP value is increased with the increase of the protein concentration. The concentration corresponding to 0.8 times of the maximum FP value is the optimal concentration of the protein. It can be seen from the figure that the interval of FP value change is most significant when the protein concentration is 1. mu.M, and the protein dosage is saved. So that GST-nNOS is finally selected_1-133The protein concentration was 1. mu.M.

FIG. 7: schematic diagram for determination of optimal incubation time. GST-nNOS_1-133After the protein is incubated for 1h, 2h, 4h, 8h and 24h, the change of FP value is basically stable, so the GST-nNOS finally selected_1-133The protein incubation time was 1 h.

FIG. 8: graph showing the effect of DMSO on FP values. The DMSO content is up to 6%, and the FP value is not affected basically.

FIG. 9: schematic diagram of FP method active primary screening.

FIG. 10: FP method screening Che-ADWV, Che-ADAV, Che-ADIV, Che-GDLV and Che-GDWV 5 polypeptides nNOS-Capon decoupling activity diagram.

FIG. 11: Che-AD (OMe) AV administration TTC staining of brain sections after rat MACO model.

FIG. 12: che-ad (ome) AV administration a histogram of infarct area of brain sections after rat MACO model.

Detailed Description

Synthesis of N-Ns-Ala-OMe

100mL of dichloromethane was added to a 250mL flask, followed by addition of methyl alanine (6g,58.2mmol) and triethylamine (16mL,116.4mmol) in that order, stirring at room temperature for 20min, addition of o-nitrobenzenesulfonyl chloride (12.9g,58.2mmol) under ice bath, stirring for 15min, removal of the ice bath, and stirring at room temperature for 2 h. Reaction ofAfter the reaction is finished, the solvent is removed by spinning, ethyl acetate is added for dissolution, and 5% of Na₂CO₃Washing for 2 times, washing with saturated salt for 2 times, collecting organic layer, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate to obtain yellow oily liquid, and performing silica gel column chromatography (PE: EA is 3:1) to obtain yellow brown solid N-Ns-Ala-OMe 15g with a yield of 89%.¹H NMR(400MHz,DMSO-d6)：8.73(s,1H),8.04–7.92(m,2H),7.89–7.82(m,2H),4.06(q,J＝7.1Hz,1H),3.46(s,3H),1.28(d,J＝7.2Hz,3H).MS(ESI)calcd for C₁₀H₁₂N₂O₆S[M+Na]⁺:311.0；found:m/z 311.0.

Synthesis of N-Che-N-Ns-Ala-OMe

200mL of redistilled tetrahydrofuran was added to a 500mL two-necked flask, and N-Ns-Ala-OMe (15g,52mmol), triphenylphosphine (20.46g,78mmol), cyclohexylethanol (7.25mL,52mmol) and Ar were added in this order, and stirring was performed in an ice bath with stirring while adding diisopropyl azodicarboxylate dropwise, and after completion of dropwise addition, the mixture was warmed to room temperature and stirred overnight. After the reaction is finished, the reaction solution is concentrated, ethyl acetate is added for dissolving, the solution is washed for 3 times, an organic layer is collected, anhydrous sodium sulfate is dried, the filtration is carried out, the filtrate is concentrated to obtain yellow oily liquid, silica gel column chromatography (PE: EA is 4:1) is carried out to obtain light yellow solid N-Che-N-Ns-Ala-OMe 15.1g, and the yield is 73%.¹H NMR(400MHz,CDCl₃):8.16–7.97(m,1H),7.72(m,2H),7.65–7.55(m,1H),4.75(q,J＝7.3Hz,1H),3.59(s,3H),3.53–3.04(m,2H),1.68–0.82(m,16H).

Synthesis of N-Che-N-Ns-Ala-OH

N-Che-N-Ns-Ala-OMe (15g,37.6mmol) was placed in a 250mL flask, dissolved in 50mL of methanol, and then added with 1N sodium hydroxide solution (45mL,45mmol) and stirred at room temperature overnight. After the reaction is finished, adding water to dissolve, adjusting the solution to 1-2 by 2N hydrochloric acid, extracting for 3 times by ethyl acetate, collecting an organic layer, drying by anhydrous sodium sulfate, filtering, and concentrating the filtrate to obtain yellow oily liquid. Silica gel column Chromatography (CH)₂Cl₂MeOH 20:1) to obtain Ac-Ala- (CSNH) -Val-OH 13.5g as orange oily liquid, which was 93% in two steps. 1H NMR (400MHz, CDCl3) 9.75(s,1H), 8.18-7.98 (m,1H),7.71(m,2H),7.64(m,1H),4.78(q, J ═ 7.3Hz,1H), 3.58-3.00 (m,2H), 1.99-0.79 (m,16H). MS (ESI) calcd for C₁₇H₂₄N₂O₆S[M+Na]⁺:407.1；found:m/z 407.1.

Synthesis of N-Che-N-Boc-Ala-OH

To a dry 250mL flask were added 50mL of redistilled N, N-dimethylformamide, N-Che-N-Ns-Ala-OH (13g, 34mmol), sodium thiophenolate (9g, 68mmol) in that order, and the mixture was stirred at room temperature overnight. After the reaction is finished, concentrating the reaction solution to a small volume by using an oil pump, adding water for dissolving, adjusting the pH to 4 by using 2N hydrochloric acid, extracting by using diethyl ether for 2 times, collecting a water layer, adjusting to 5-6 by using 1N sodium hydroxide solution, and freeze-drying to obtain a light yellow solid N-Che-Ala-OH crude product. MS (ESI) calcd for C₁₁H₂₁NO₂[M+Na]⁺:222.1；found:m/z 222.1.

To the above solid was added triethylamine (19mL,136mmol), (Boc)₂O (22.3g, 102mmol), and stirred at room temperature overnight. After the reaction is finished, spin-drying the solvent, dissolving in water, adjusting to 3-4 with 10% citric acid, extracting with ethyl acetate for three times, collecting organic layer, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate to obtain yellow oily liquid, and performing silica gel column Chromatography (CH)₂Cl₂MeOH ═ 20:1) gave 5.1g of N-Che-N-Boc-Ala-OH as a clear oily liquid in 50% yield over two steps.¹H NMR(400MHz,CDCl₃):11.31(s,1H),4.42-3.99(m,1H),3.36-3.06(m,2H),1.81–0.84(m,26H).MS(ESI)calcd forC₁₂H₂₉NO₄[M+Na]⁺:322.2；found:m/z 322.2.

The tetrapeptides described in the present invention were then synthesized by conventional solid phase synthesis according to the scheme of FIG. 2.