CN111533780A - Polypeptide with nNOS-Capon uncoupling activity and application thereof - Google Patents

Abstract

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-Xaa¹‑Xaa²‑Xaa³-Val, wherein Che = N-Cyclohexylethyl, Xaa¹= Ala or Gly, Xaa²= Glu, Glu (OMe), Asp or Asp (OMe), Xaa³= 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).

Description

Polypeptide with nNOS-Capon uncoupling activity and application thereof

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.

Example 1N-Cyclohexylethyl-Ala-Glu-Ala-Val (Che-AEAV)

After activating dried 2-chlorotrimethyl chloride resin (load rate 1.056mmol), taking Fmoc-Val-OH as a starting material, repeating Fmoc removal and amino acid condensation reaction for multiple times, wherein the specific synthetic route is shown in figure 2, and obtaining Che-AEAV155.5mg with the yield of 15.61%. HPLC t_R＝13.45min.MS(ESI)calcd for C₂₄H₄₂N₄O₇[M-H]^-:497.3；found:m/z 497.3.¹H NMR(300MHz,DMSO-d₆)12.30(s,2H),8.64(d,J＝8.3Hz,1H),8.07(d,J＝7.1Hz,1H),7.90(d,J＝8.6Hz,1H),4.34(t,J＝7.0Hz,2H),4.10(dd,J＝8.5,5.7Hz,1H),3.89(s,1H),2.78(s,3H),2.24(t,J＝8.0Hz,2H),2.01–1.89(m,1H),1.87(d,J＝5.2Hz,1H),1.83–1.68(m,1H),1.61–1.47(m,5H),1.42(t,J＝7.6Hz,2H),1.33(d,J＝6.8Hz,3H),1.18(d,J＝7.0Hz,6H),0.84(d,J＝6.8Hz,9H).

Example 2N-Cyclohexylethyl-Ala-Glu-Trp-Val (Che-AEWV)

Che-AEWV 76.3mg was obtained in 12.45% yield according to the synthesis in example 1. HPLC t_R＝14.15min.MS(ESI)calcd for C₃₂H₄₇N₅O₇[M-H]^-:612.3；found:m/z 612.4.¹H NMR(300MHz,DMSO-d₆)13.04–11.60(m,2H),10.75(s,1H),8.58(d,J＝8.5Hz,1H),8.08(t,J＝8.5Hz,2H),7.82–6.38(m,5H),4.65(d,J＝4.9Hz,1H),4.33(d,J＝5.4Hz,1H),4.21–4.06(m,1H),3.81(d,J＝7.0Hz,1H),3.09(d,J＝10.0Hz,2H),2.97–2.63(m,3H),2.15–2.05(m,3H),2.06(s,1H),1.87(s,1H),1.73(d,J＝7.9Hz,1H),1.60(d,J＝9.5Hz,5H),1.55–1.32(m,3H),1.23–1.12(m,4H),1.13(s,3H),0.87–0.71(m,6H).

Example 3N-Cyclohexylethyl-Ala-Asp-Trp-Val (Che-ADWV)

Che-ADWV 38.4mg was obtained in 6.41% yield according to the synthesis in example 1. HPLC t_R＝14.35min.MS(ESI)calcd for C₃₁H₄₅N₅O₇[M-H]^-:598.3；found:m/z 598.3.¹H NMR(300MHz,DMSO-d₆)10.75(s,2H),8.66(s,1H),7.96(s,2H),7.55(d,J＝7.8Hz,1H),7.28(d,J＝8.4Hz,1H),7.13–6.77(m,4H),4.58(s,2H),4.12(s,1H),3.68(s,1H),3.08(s,2H),2.93(s,2H),2.67–2.35(m,3H),2.01(s,1H),1.58(s,5H),1.38(d,J＝7.5Hz,3H),1.23(d,J＝6.8Hz,5H),1.12–0.99(m,3H),0.84(d,J＝4.1Hz,6H).

Example 4N-Cyclohexylethyl-Ala-Glu-Phe-Val (Che-AEFV)

Che-AEFV 97.8mg was obtained according to the synthesis in example 1, with a yield of 17.03%. HPLC t_R＝10.17min.MS(ESI)calcd for C₃₀H₄₆N₄O₇[M-H]^-:573.3；found:m/z 573.3.¹H NMR(300MHz,DMSO-d₆)8.56(d,J＝8.0Hz,1H),8.12–8.00(m,2H),7.26–7.07(m,5H),4.62(s,1H),4.29(d,J＝5.2Hz,1H),4.12(d,J＝8.4Hz,1H),3.80(d,J＝7.2Hz,1H),2.99–2.90(m,2H),2.85–2.65(m,3H),2.16(t,J＝8.0Hz,2H),2.03–1.89(m,2H),1.83(s,1H),1.77–1.44(m,7H),1.42–1.17(m,6H),1.13(s,3H),0.89–0.80(m,6H).

Example 5N-Cyclohexylethyl-Ala-Asp-Ala-Val (Che-ADAV)

Che-ADAV 33.2mg was obtained in 6.86% yield according to the synthesis in example 1. HPLC t_R＝11.18min.MS(ESI)calcd for C₂₃H₄₀N₄O₇[M-H]^-:483.3；found:m/z 483.2.¹H NMR(300MHz,DMSO-d₆)12.47(s,2H),9.09–8.73(m,1H),7.99(d,J＝6.9Hz,1H),7.90(s,1H),4.63(s,1H),4.44–4.27(m,1H),4.22–4.03(m,1H),3.81(s,1H),2.80(s,2H),2.73–2.50(m,2H),2.52(d,J＝9.6Hz,1H),2.10–1.94(m,1H),1.63–1.42(m,5H),1.44(d,J＝6.7Hz,2H),1.36(d,J＝6.8Hz,3H),1.19(d,J＝7.0Hz,6H),0.87(s,3H),0.85(s,6H).

Example 6N-Cyclohexylethyl-Ala-Glu-Ile-Val (Che-AEIV)

Che-AEIV 23.5mg was obtained in 4.35% yield according to the synthesis in example 1. HPLC t_R＝12.95min.MS(ESI)calcd for C₂₇H₄₈N₄O₇[M-H]^-:539.3；found:m/z 539.3.¹H NMR(300MHz,DMSO-d₆)8.54(s,1H),7.92(d,J＝8.5Hz,1H),7.85(s,1H),4.40(s,1H),4.33–4.21(m,1H),4.14–4.05(m,1H),3.73(s,1H),2.75–2.27(m,3H),2.20(d,J＝7.5Hz,3H),2.13–1.96(m,2H),1.88(s,1H),1.74(s,2H),1.63–1.51(m,5H),1.50–1.36(m,4H),1.35–1.10(m,7H),0.87(d,J＝2.7Hz,3H),0.83(d,J＝7.3Hz,6H),0.78(d,J＝7.4Hz,3H).

Example 7N-Cyclohexylethyl-Ala-Asp-Ile-Val (Che-ADIV)

Che-ADIV 67.5mg was obtained in 12.82% yield according to the synthesis in example 1. HPLC t_R＝10.90min.MS(ESI)calcd for C₂₆H₄₆N₄O₇[M+H]⁺:527.3；found:m/z 527.4.¹H NMR(300MHz,DMSO-d₆)8.75(d,J＝7.9Hz,1H),7.94(d,J＝8.3Hz,1H),7.71(d,J＝9.3Hz,1H),4.65(d,J＝4.8Hz,1H),4.33–4.21(m,1H),4.14–4.02(m,1H),3.71(d,J＝6.7Hz,1H),2.73–2.61(m,3H),2.66(d,J＝4.5Hz,1H),2.52–2.37(m,2H),2.03–1.87(m,1H),1.63-1.50(m,6H),1.48–1.35(m,3H),1.31(d,J＝6.7Hz,4H),1.17–0.98(m,3H),0.91(s,2H),0.87(d,J＝2.5Hz,3H),0.84(d,J＝4.9Hz,6H),0.77(d,J＝7.4Hz,3H).

Example 8N-Cyclohexylethyl-Gly-Asp-Ala-Val (Che-GDAV)

Che-GDAV 248.6mg was obtained in 26.42% yield according to the synthesis in example 1. HPLC t_R＝16.50min.MS(ESI)calcd for C₂₂H₃₈N₄O₇[M-H]^-:469.3；found:m/z 469.3.¹H NMR(300MHz,DMSO-d₆)12.59(s,2H),8.73–8.61(m,1H),8.10(d,J＝7.0Hz,1H),7.88–7.72(m,1H),4.69–4.53(m,1H),4.43–4.26(m,1H),4.15–4.04(m,1H),3.70(s,2H),2.98–2.81(m,2H),2.69–2.67(m,1H),2.61–2.50(m,2H),2.04–1.89(m,1H),1.63–1.41(m,5H),1.45–1.23(m,2H),1.19(t,J＝6.3Hz,4H),1.00–0.93(m,3H),0.95(s,2H),0.85(s,6H).

Example 9N-Cyclohexylethyl-Gly-Asp-Pro-Val (Che-GDPV)

The synthesis in example 1 gave 176.3mg of Che-GDPV, 17.78% yield. HPLC t_R＝14.17min.MS(ESI)calcd for C₂₄H₄₀N₄O₇[M-H]^-:495.3；found:m/z 495.3.¹H NMR(300MHz,DMSO-d₆)12.51(s,2H),8.90(d,J＝7.7Hz,1H),7.81(d,J＝8.5Hz,1H),4.87(d,J＝5.4Hz,1H),4.40(d,J＝6.3Hz,1H),4.12–4.03(m,1H),3.69(s,2H),3.65–3.54(m,2H),2.90(s,2H),2.72–2.66(m,1H),2.50(s,2H),2.47–2.38(m,2H),2.04–1.97(m,1H),1.89(s,2H),1.54–1.31(m,7H),1.30–1.08(m,4H),0.92(d,J＝7.0Hz,2H),0.86(d,J＝6.8Hz,6H).

Example 10N-Cyclohexylethyl-Gly-Asp-Leu-Val (Che-GDLV)

Che-GDLV 318mg was obtained in 31.06% yield according to the synthesis in example 1. HPLC t_R＝13.90min.MS(ESI)calcd for C₂₅H₄₄N₄O₇[M-H]^-:511.3；found:m/z 511.3.¹H NMR(300MHz,DMSO-d₆)8.75(d,J＝7.9Hz,1H),8.09(d,J＝8.1Hz,1H),7.80(d,J＝8.4Hz,1H),4.64–4.58(m,1H),4.34–4.27(m,1H),4.13–4.00(m,1H),3.77–3.56(m,2H),2.90(s,2H),2.75–2.58(m,1H),2.50(d,J＝9.5Hz,2H),2.02–1.87(m,1H),1.64(s,2H),1.61(s,4H),1.46–1.38(m,4H),1.28–1.04(m,4H),0.89(d,J＝9.2Hz,2H),0.87–0.78(m,12H).

Example 11N-Cyclohexylethyl-Gly-Asp-Phe-Val (Che-GDFV)

Che-GDFV 151.5mg was obtained in 13.74% yield according to the synthesis in example 1. HPLC t_R＝14.15min.MS(ESI)calcd for C₂₈H₄₂N₄O₇[M+H]⁺:547.3；found:m/z 547.3.¹H NMR(300MHz,DMSO-d₆)12.62(s,2H),8.69–8.43(m,1H),8.22–7.93(m,2H),7.22–6.89(m,5H),4.72–4.41(m,2H),4.13(dd,J＝8.2,6.0Hz,1H),3.62–3.54(m,2H),3.43(s,2H),3.08–2.95(m,1H),2.67–2.59(m,2H),2.50–2.36(m,2H),2.05–1.78(m,1H),1.53–1.44(m,7H),1.31–1.00(m,4H),0.94–0.74(m,8H).

Example 12N-Cyclohexylethyl-Gly-Asp-Trp-Val (Che-GDWV)

Che-GDWV 189.1mg was obtained in 16.16% yield according to the synthesis in example 1. HPLC t_R＝14.10min.MS(ESI)calcd for C₂₅H₄₄N₄O₇[M+H]⁺:586.3；found:m/z 586.3.¹H NMR(300MHz,DMSO-d₆)10.78(d,J＝8.8Hz,1H),8.67–8.57(m,1H),8.16–7.94(m,2H),7.57(d,J＝6.4Hz,1H),7.30(d,J＝7.9Hz,1H),7.13–6.91(m,3H),4.69–4.52(m,2H),4.12(d,J＝8.8Hz,1H),3.70–3.53(m,2H),3.12–3.01(m,2H),2.98–2.80(m,3H),2.76–2.50(m,2H),2.04–1.66(m,1H),1.56–1.44(m,6H),1.42(s,1H),1.27–1.07(m,4H),0.87(d,J＝6.4Hz,8H).

Example 13N-Cyclohexylethyl-Ala-Asp (OMe) -Ala-Val (Che-AD (OMe) AV)

Che-AD (OMe) AV 88mg was obtained according to the synthesis in example 1, in 16.7% yield. HPLC t_R＝20min.¹HNMR(400MHz,)8.88(d,J＝8.0Hz,1H),8.10(d,J＝8.9Hz,1H),7.89(d,J＝9.6Hz,1H),4.67-4.62(m,1H),4.34-4.27(m,1H),4.10-4.06(m,1H),3.82-3.77(m,1H),3.55(s,3H),2.87–2.49(m,5H),2.08–1.92(m,1H),1.62-1.55(m,5H),1.44-1.38(m,2H),1.32(d,J＝6.9Hz,3H),1.25–1.04(m,9H),0.82(d,J＝6.7Hz,6H).MS(ESI,m/z):499.3[M+H]⁺

EXAMPLE 14 establishment of method for measuring nNOS-Capon by fluorescence polarization method

1.GST-nNOS_1-133Induced expression and purification of proteins

Will contain pGEX4T-1-GST-nNOS_1-133E.coli BL21(DE3) was inoculated into 5mL of LB medium containing 100. mu.g/mL ampicillin and cultured overnight on a shaker at 37 ℃ and 180 r/min. The next day, the cells were transferred to 300mL of LB medium containing 100. mu.g/mL of ampicillin and cultured for 2.5 hours, and then induced by addition of lactose to a final concentration of 2.5g/mL, and the culture was continued for 6 hours. Centrifuging at 4 deg.C for 30min at 3000g, collecting precipitate, suspending in cold (concentration: 10mM, 7.4), adding lysozyme, shaking, mixing, and placing in a refrigerator at-20 deg.C overnight. The next day, the bacterial solution was re-melted in a shaker at 37 ℃ and 180r/min, and then the genomic DNA was digested by the addition of DNase. Centrifugation was carried out at 15000rpm for 15min at 4 ℃ and the supernatant was collected and filtered through a 0.22 μm microfiltration membrane. Adding the collected supernatant into a GST affinity chromatography column, eluting with reduced glutathione (0.154g/50mL PBS), and purifying to obtain GST-nNOS_1-133And (3) carrying out ultrafiltration concentration on the protein in a 30KDa ultrafiltration tube, and determining the protein concentration by using a BCA kit.

2. Identification of GST-nNOS by Coomassie brilliant blue staining method_1-133Protein

SDS-PAGE electrophoresis is carried out on the purified and concentrated protein, Coomassie brilliant blue staining solution is added for staining overnight, destaining solution is adopted for eluting the next day, white light shooting is carried out, the result is shown in figure 3, and expressed GST-nNOS_1-133High correctness and purity.

Isothermal Titration Calorimetry (ITC) was used to fit fluorescent molecular probes 5-FAM-KV-14 to GST-nNOS_1-133And (3) a protein binding process, namely verifying that the protein is combined into a typical single-site binding model and meets the screening requirement of a fluorescence polarization method. As shown in FIG. 4, binding of probe to protein at saturationThe concentration ratio is about 10:1, which provides a reference for the subsequent selection of the concentration of fluorescent molecular probes and proteins.

3. Determination of optimal fluorescent molecular probe concentration

And (3) determining the influence of the fluorescent molecular probe 5-FAM-KV-14 with different concentrations on the FP value. Adding 5-FAM-KV-14 standard solution (500 mu M) and 1xHepes into a 96-well plate according to the calculated volume respectively, enabling the final concentration of the fluorescent molecular probe 5-FAM-KV-14 in each well to be the concentration described above, controlling the total volume of each well to be 100 mu L, setting 3 parallel wells for each concentration, measuring the FP value by using a multifunctional microplate reader at 37 ℃, and drawing by using statistical software Graphpad Prism 7.

As shown in FIG. 5, in the probe well containing only free fluorescent molecules, the FP value tended to decrease with increasing concentration of 5-FAM-KV-14. At 50nM 5-FAM-KV-14, a FP value of 57.25mP was detected and stabilized with increasing probe concentration. Thus, the final concentration of 5-FAM-KV-14 was 50 nM.

4. Optimum GST-nNOS_1-133Determination of protein concentration

In the case of an optimum fluorescent molecular probe concentration (50nM) determined, GST-nNOS at different concentrations were determined_1-133Effect of protein on FP values. 5-FAM-KV-14 standard solution (500. mu.M) and 1xHepes were added to the 96-well plate in the calculated volume, respectively, so that the final concentration of 5-FAM-KV-14 in the well was 50 nM. Followed by addition of a calculated volume of GST-nNOS_1-133The final concentration of the protein stock solution (93.02. mu.M) in each well was set to the above-described concentration, the total volume of each well was controlled to 125. mu.L, 3 wells were set in each concentration, and FP values were measured by a multifunctional microplate reader at 37 ℃ and plotted by the statistical software GraphpadPrism 7.

As shown in FIG. 6, 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. Thus, GST-nNOS is finally selected_1-133The protein concentration was 1. mu.M.

5. Determination of optimum incubation time

Selection of 5-FAM-KV-14 and GST-nNOS_1-133The optimal incubation time of the protein can make the detected FP value stronger and more stable, thus being beneficial to reducing errors caused by environmental factors and improving the accuracy of the screening method.

5-FAM-KV-14 (final concentration: 50nM) and GST-nNOS in different concentrations were added to a 96-well plate_1-133The total volume of the protein and 1xHepes in each hole is controlled to be 125 mu L, the incubation is carried out for 1h, 2h, 4h, 8h and 24h at 37 ℃, and three parallel holes are arranged at different reaction times. FP values were determined separately with a multifunctional microplate reader after incubation for different periods at 37 ℃ and plotted by statistical software Graphpad Prism 7.

As shown in FIG. 7, GST-nNOS_1-133After the protein is incubated for 1h, 2h, 4h, 8h and 24h, the change of FP value is basically stable, so that GST-nNOS finally selected_1-133The protein incubation time was 1 h.

Effect of DMSO on FP values

Since some compounds have poor solubility and require a small amount of DMSO, it is necessary to determine the effect of different amounts of DMSO on FP.

5-FAM-KV-14 (final concentration: 50nM), GST-nNOS were added to a 96-well plate_1-133Protein (final concentration: 1. mu.M), 1xHepes and different volumes of DMSO, so that the DMSO content respectively accounts for 0%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 12% of the total volume, and the total volume of each well is controlled at 125. mu.L. FP values were determined separately with a multifunctional microplate reader after incubation for different periods at 37 ℃ and plotted by statistical software Graphpad Prism 7.

As shown in FIG. 8, the DMSO content is up to 6%, which has no effect on the FP value, and when the DMSO content is up to 6%, the FP value is lowered. Therefore, the DMSO content is selected to be only up to 6%.

7. Reliability evaluation method adopting Z factor and signal-to-noise ratio

After determination of the conditions such as concentration and incubation time, 3 parallel wells were set in a 96-well plate, and the presence or absence of GST-nNOS was determined_1-133Protein (1. mu.M) reacted with 5-FAM-KV-14(50nM) for 1h fluorescence polarization (FP, mP). According to the following formulaCalculating the Z' value and the signal-to-noise ratio (S/N) of the fluorescence polarization system, and evaluating the reliability of the system.

Z’＝1–(3σ_binding+3σ_free)/|μ_binding-μ_free|；S/N＝(μ_binding-μ_free)/σ_free(where σ is the standard deviation and μ is the mean value, binding: 5-FAM-KV-14+ GST-nNOS_1-133FP value of (1), free: FP value of free 5-FAM-KV-14)

Finally, the average value of the Z factor of the method is 0.96, and the average value of the signal-to-noise ratio is 140.9. The fluorescent polarization screening method established in the experiment has the advantages of sensitivity, reliability and the like, and can be better used for screening nNOS-CAPON pathway small molecule inhibitors.

8. Screening of Compounds

Fluorescent probe molecule 5-FAM-KV-14 solution (final concentration: 50nM), GST-nNOS in calculated volume were added to a 96-well plate_1-133Protein solution (final concentration: 1. mu.M) and small molecule inhibitor (final concentration: 1mM), each concentration was set to 3 replicates, the total competitive inhibition volume was controlled at 125. mu.L, FP values were determined after incubation at 37 ℃ for 1h after mixing, and the preliminary screening results are shown in FIG. 9.

The further accurate test results are shown in fig. 10, and 5 compounds with binding activity are obtained by screening the synthesized target compounds through a competitive fluorescence polarization experiment, and can be used as novel tetrapeptide molecular probes for further protein binding research.

Example 15 transient cerebral ischemia model

The experimental protocol was approved by the animal care and use committee of the university of medical, Nanjing. All efforts were made to reduce the number of rats used and their suffering. In this study we used Sprague-Dawley rats (9-10 weeks old, 250-270g) purchased from the animal center of university of Nantong. Food and water were taken ad libitum with maintaining room temperature 22 + -2 deg.C and light (12 hours light and shade cycle). Animals were randomly assigned to experimental groups by computer. The experimenter marks all animals according to the randomization schedule before assignment. The experiments were performed by researchers who did not know to which group the animals belong.

The experimental protocol was referenced to a model of transient cerebral ischemia in Zhou L et al. Briefly, SD rats were anesthetized with chloral hydrate (350mg/kg, ip) and 4/0 surgical nylon monofilament circular tips were introduced into the left internal carotid artery through the external carotid stump, advancing 20-21mm at the carotid bifurcation until slight resistance was felt. At this point, the intraluminal filaments occlude all blood sources of the middle, internal, anterior and posterior cerebral arteries. Body temperature was maintained at 37. + -. 0.5 ℃ throughout the procedure. The tether was left in place for 120min and then removed for reperfusion. Immediately after removal of the plug wire, administration was via the tail vein. In sham animals, the occlusive filament was inserted only 7mm above the carotid bifurcation.

Evaluation results

Infarct volume measurements were performed 24 hours after MACO. Brains were removed quickly and frozen at-20 ℃ for 5 min. 1-2 mm coronal sections were prepared and immersed in 2% TTC at 37 ℃ for 4 h. The staining results are shown in FIGS. 11 and 12, and infarct volume is expressed as the percentage area of the coronal section in the infarcted hemisphere. Model group 24.6%, Che-AD (OMe) AV 4.5%, edaravone group 13.5%.

Claims (5)

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.

Images