CN111533780B - Polypeptide with nNOS-Capon uncoupling activity and application thereof - Google Patents

Polypeptide with nNOS-Capon uncoupling activity and application thereof Download PDF

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CN111533780B
CN111533780B CN202010296229.XA CN202010296229A CN111533780B CN 111533780 B CN111533780 B CN 111533780B CN 202010296229 A CN202010296229 A CN 202010296229A CN 111533780 B CN111533780 B CN 111533780B
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CN111533780A (en
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秦亚娟
厉廷有
冯玲玲
徐剑
郑礼平
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Nanjing Medical University
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Nanjing Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1008—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Abstract

A polypeptide with nNOS-Capon uncoupling activity and its application are disclosed, which are characterized in that the structural general formula of amino acid sequence is CheXaa 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. The polypeptide has nNOS-Capon decoupling activity and can be used as a neuroprotectant for treating cerebral arterial thrombosis. A fluorescence polarization method (FP) for in vitro rapid screening of nNOS-Capon uncoupling activity is provided. The polypeptide screened by the FP method has obvious neuroprotection in 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 cerebral apoplexy is one of the most important disability and death diseases in the world, seriously harms human health, and the development of therapeutic drugs is one of the most important subjects of pharmaceutical chemistry. At present, the research of cerebral apoplexy drugs mainly comprises: calcium channel antagonists, glutamate release inhibitors, GABA receptor agonists, nNOS inhibitors, free radical scavengers, MMP-9 inhibitors, NMDAR antagonists, etc., but the therapeutic effect is not ideal. Many neuroprotective agents effective in animal models of cerebral stroke have failed to achieve the desired therapeutic effect in human clinical trials or have to terminate clinical trials due to too great side effects.
When ischemic stroke occurs, the toxic effects of Excitatory Amino Acids (EAA) play an important role in the pathogenesis of ischemic stroke. Studies have shown that the NMDAR-PSD95-nNOS signaling pathway mediates the toxic effects of stroke excitatory glutamate. However, since NMDAR and nNOS mediate many important physiological functions, inhibiting their activity can produce many toxic and side effects. For example, clinical studies have shown that NMDAR antagonists cannot be developed into therapeutic drugs due to their side effects. In excitotoxic stimulation, a stimulation signal is transmitted to nNOS through NMDAR-PSD95-nNOS, nNOS is coupled with Capon, and Capon can be coupled with MKK3 to form an nNOS-Capon-MKK3 ternary complex. nNOS can activate MKK3, which in turn activates the p38MAPK signaling pathway, resulting in neuronal cell death. Studies have shown that inhibition of nNOS-Capon coupling has neuroprotective effects.
The nNOS PDZ domain contains 127 amino acid residues and Capon is the natural ligand for nNOS. Capon binds between the αb helix and βb sheet of nNOS PDZ domain via its carboxy-terminal tetrapeptide EIAV, which is a shallow and long groove containing a binding pocket consisting of the conserved sequences GLGF (Gly 21, leu 22, gly23, phe 24). Polypeptides Che-a/G-D/E-X-V and Che-a/G-D/E (OMe) -X-V designed according to the carboxy-terminal EIAV of Capon act as nNOS-Capon uncoupling by competitively binding to the GLGF domain pocket. The nNOS-Capon uncoupling activity of the polypeptide is screened and tested by using a fluorescence polarization method, the compound with good activity is screened out, the structure of the compound is optimized by esterification, the drug property is improved, and the neuroprotective activity of the compound is tested by using a MACO animal model.
Disclosure of Invention
The technical problems to be solved are as follows: the invention provides a polypeptide with nNOS-Capon uncoupling activity and application thereof, and the polypeptide can be used as a neuroprotectant for treating ischemic cerebral apoplexy.
The technical scheme is as follows: polypeptide with nNOS-Capon uncoupling activity and amino acid sequence structural general formula of CheXaa 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.
The structural formula is any one of the following compounds:
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 polypeptide structures are as follows:
the amino acid of the invention adopts an amino acid fragment modification method to combine with a traditional solid phase synthesis method, and the synthesis route is shown in figures 1 and 2.
The application of the polypeptide or the pharmaceutically acceptable salt thereof in preparing neuroprotective medicines.
The application of the polypeptide or the pharmaceutically acceptable salt thereof in preparing medicaments for treating stroke, anxiety or depression.
A neuroprotective agent contains the above polypeptide or pharmaceutically acceptable salt thereof as effective component.
The beneficial effects are that: the polypeptide provided by the invention has obvious neuroprotection in a rat cerebral ischemia reperfusion Model (MACO), wherein the area of cerebral infarction of a MACO model brain slice TTC staining model group is 24.6%, the area of Che-AD (OMe) AV cerebral infarction is 4.5%, and the area of edaravone cerebral infarction of a positive control group is 13.5%.
Drawings
FIG. 1 is a synthetic diagram of alkylated amino acids;
FIG. 2 is a synthetic diagram of alkylated tetrapeptides;
fig. 3: GST-nNOS 1-133 A western blot identification chart. Expression purification of the concentrated protein SDS-PAGE electrophoresis, followed by coomassie brilliant blue staining to identify GST-nNOS 1-133 。
Fig. 4:5-FAM-KV-14 and GST-nNOS 1-133 Schematic of the binding ITC. Fitting fluorescent molecular probes 5-FAM-KV-14 and GST-nNOS by isothermal titration calorimetry (isothermal titration calorimetry, ITC) 1-133 And in the binding process of the protein, the binding is verified to be a typical single-point binding model, and the screening requirement of a fluorescence polarization method is met.
Fig. 5: schematic of the concentration of the optimal fluorescent molecular probe. With the increase of the concentration of 5-FAM-KV-14, the FP value tends to be reduced. When 5-FAM-KV-14 was 50nM, the FP value detected at this time was 57.25mP, and as the probe concentration increased, the FP value tended to be stable, so that the final 5-FAM-KV-14 concentration was 50nM.
Fig. 6: optimum GST-nNOS 1-133 Schematic of the determination of protein concentration. With GST-nNOS 1-133 The FP values tended to increase with increasing protein concentration. The concentration corresponding to 0.8 times of the maximum FP value is the optimal concentration of the protein. As can be seen from the graph, the FP value change interval is most pronounced at a protein concentration of 1. Mu.M, and the protein amount is also relatively low. Therefore, the GST-nNOS is finally selected 1-133 The protein concentration was 1. Mu.M.
Fig. 7: schematic of determination of optimal incubation time. GST-nNOS 1-133 After 1h, 2h, 4h, 8h and 24h incubation of the proteinThe FP value is basically stable, so that the GST-nNOS which is finally selected 1-133 The protein incubation time was 1h.
Fig. 8: the effect of DMSO on FP values is schematically indicated. The DMSO content is up to 6% and has no substantial effect on the FP value.
Fig. 9: schematic of FP method activity primary screening.
Fig. 10: the FP assay screens nNOS-Capon decoupling activity of 5 polypeptides, cheADWV, cheADV, cheADIV, cheGDLV, cheGDWV.
Fig. 11: brain slice TTC staining pattern after administration of the rat MACO model by CheAD (OMe) AV.
Fig. 12: brain slice infarct size histogram after administration of Che-AD (OMe) AV to rat MACO model.
Detailed Description
Synthesis of N-Ns-Ala-OMe
To a 250mL flask was added 100mL of methylene chloride followed by alanine methyl ester (6 g,58.2 mmol), triethylamine (16 mL,116.4 mmol), stirred at room temperature for 20min, o-nitrobenzenesulfonyl chloride (12.9 g,58.2 mmol) was added under ice-bath conditions, stirred for 15min, the ice-bath was removed, and stirred at room temperature for 2h. After the reaction, the solvent was removed by spinning, ethyl acetate was added to dissolve, 5% Na 2 CO 3 Washing for 2 times, washing with saturated saline for 2 times, collecting organic layer, drying with anhydrous sodium sulfate, filtering, concentrating filtrate to obtain yellow oily liquid, and performing silica gel column chromatography (PE: EA=3:1) to obtain yellow brown solid N-Ns-Ala-OMe 15g with 89% yield. 1 H NMR(400MHz,DMSO-d6)δ：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 C 10 H 12 N 2 O 6 S[M+Na] + :311.0；found:m/z 311.0.
Synthesis of N-CheN-Ns-Ala-OMe
200mL of redistilled tetrahydrofuran was added to a 500mL double-necked flask, followed by N-Ns-Ala-OMe (15 g,52 mmol), triphenylphosphine (20.46 g,78 mmol), cyclohexylethanol (7.25 mL,52 mmol), ar protected, stirred under ice-bath conditions, diisopropyl azodicarboxylate was added dropwise with stirring, and after the dropwise addition was completed, the mixture was warmed to room temperature and stirred overnight. After the reaction, the reaction solution is concentrated and added with the ethyl acetateDissolving ethyl acetate, washing for 3 times, collecting an organic layer, drying with anhydrous sodium sulfate, filtering, concentrating the filtrate to obtain yellow oily liquid, and performing silica gel column chromatography (PE: EA=4:1) to obtain 15.1g of light yellow solid N-CheN-Ns-Ala-OMe with the yield of 73%. 1 H NMR(400MHz,CDCl 3 )δ: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-CheN-Ns-Ala-OH
N-Chen-N-Ns-Ala-OMe (15 g,37.6 mmol) was taken in a 250mL flask, dissolved in 50mL methanol, added with 1N sodium hydroxide solution (45 mL,45 mmol) and stirred overnight at room temperature. After the reaction, water was added to dissolve, 2N hydrochloric acid was adjusted to 1-2, extraction was performed 3 times with ethyl acetate, the organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to obtain a yellow oily liquid. Silica gel column Chromatography (CH) 2 Cl 2 Meoh=20:1) to give 13.5g of Ac-Ala- (CSNH) -Val-OH as an orange yellow oil in 93% yield in two steps. 1H NMR (400 MHz, CDCl 3) delta 9.75 (s, 1H), 8.18-7.98 (m, 1H), 7.71 (m, 2H), 7.64 (m, 1H), 4.78 (q, J=7.3 Hz, 1H), 3.58-3.00 (m, 2H), 1.99-0.79 (m, 16H) MS (ESI) calcd for C 17 H 24 N 2 O 6 S[M+Na] + :407.1；found:m/z 407.1.
Synthesis of N-CheN-Boc-Ala-OH
To a dry 250mL flask was added sequentially 50mL of redistilled N, N-dimethylformamide, N-Chen-N-Ns-Ala-OH (13 g,34 mmol), sodium thiophenol (9 g,68 mmol) and stirred overnight at room temperature. After the reaction is finished, the reaction solution is concentrated to a small volume by an oil pump, water is added for dissolution, the PH is regulated to 4 by using 2N hydrochloric acid, the diethyl ether is extracted for 2 times, a water layer is collected, the pH is regulated to 5-6 by using 1N sodium hydroxide solution, and the crude product of the light yellow solid N-CheAla-OH is obtained by freeze drying. MS (ESI) calcd for C 11 H 21 NO 2 [M+Na] + :222.1；found:m/z 222.1.
Triethylamine (19 mL,136 mmol), (Boc) was added to the solid 2 O (22.3 g,102 mmol) was stirred overnight at room temperature. After the reaction, spin-drying the solvent, adding water for dissolution, adjusting to 3-4 with 10% citric acid, extracting with ethyl acetate for three times, collecting the organic layer, drying with anhydrous sodium sulfate, filtering,concentrating the filtrate to obtain yellow oily liquid, and subjecting to silica gel column chromatography (CH 2 Cl 2 MeOH=20:1) to give a clear oily solution N-Chen-N-Boc-Ala-OH 5.1g in 50% yield in two steps. 1 H NMR(400MHz,CDCl 3 )δ: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 for C 12 H 29 NO 4 [M+Na] + :322.2；found:m/z 322.2.
The tetrapeptides described in the present invention are then synthesized using conventional solid phase synthesis according to the scheme of FIG. 2.
Example 1N-Cyclohexylethyl-Ala-Glu-Ala-Val (Che-AEAV)
After activation of the dried 2-chlorotrimethylene chloride resin (loading rate 1.056 mmol), fmoc-Val-OH was used as starting material, fmoc removal and amino acid condensation reaction was repeated several times, and the specific synthetic route was as shown in FIG. 2, giving CheAEAV 155.5mg with a yield of 15.61%. HPLC: t R ＝13.45min.MS(ESI)calcd for C 24 H 42 N 4 O 7 [M-H] - :497.3；found:m/z 497.3. 1 H NMR(300MHz,DMSO-d 6 )δ12.30(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)
According to the synthesis method in example 1, che-AEWV 76.3mg was obtained in a yield of 12.45%. HPLC: t R ＝14.15min.MS(ESI)calcd for C 32 H 47 N 5 O 7 [M-H] - :612.3；found:m/z 612.4. 1 H NMR(300MHz,DMSO-d 6 )δ13.04–11.60(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)
According to the synthesis method in example 1, cheADWV 38.4mg was obtained in a yield of 6.41%. HPLC: t R ＝14.35min.MS(ESI)calcd for C 31 H 45 N 5 O 7 [M-H] - :598.3；found:m/z 598.3. 1 H NMR(300MHz,DMSO-d 6 )δ10.75(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)
97.8mg of Che-AEFV was obtained in the same manner as in example 1 with a yield of 17.03%. HPLC: t R ＝10.17min.MS(ESI)calcd for C 30 H 46 N 4 O 7 [M-H] - :573.3；found:m/z 573.3. 1 H NMR(300MHz,DMSO-d 6 )δ8.56(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 (CheADAV)
33.2mg of CheADAV was obtained in the same manner as in example 1, with a yield of 6.86%. HPLC: t R ＝11.18min.MS(ESI)calcd for C 23 H 40 N 4 O 7 [M-H] - :483.3；found:m/z 483.2. 1 H NMR(300MHz,DMSO-d 6 )δ12.47(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)
23.5mg of CheAEIV was obtained according to the synthesis procedure in example 1, giving a yield of 4.35%. HPLC: t R ＝12.95min.MS(ESI)calcd for C 27 H 48 N 4 O 7 [M-H] - :539.3；found:m/z 539.3. 1 H NMR(300MHz,DMSO-d 6 )δ8.54(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)
67.5mg of CheADIV was obtained according to the synthesis method in example 1 in a yield of 12.82%. HPLC: t R ＝10.90min.MS(ESI)calcd for C 26 H 46 N 4 O 7 [M+H] + :527.3；found:m/z 527.4. 1 H NMR(300MHz,DMSO-d 6 )δ8.75(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)
According to the synthesis method in example 1, 248.6mg of CheGDAV was obtained in 26.42% yield. HPLC: t R ＝16.50min.MS(ESI)calcd for C 22 H 38 N 4 O 7 [M-H] - :469.3；found:m/z 469.3. 1 H NMR(300MHz,DMSO-d 6 )δ12.59(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)
176.3mg of CheGDPV were obtained in accordance with the synthesis procedure as in example 1 with a yield of 17.78%. HPLC: t R ＝14.17min.MS(ESI)calcd for C 24 H 40 N 4 O 7 [M-H] - :495.3；found:m/z 495.3. 1 H NMR(300MHz,DMSO-d 6 )δ12.51(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)
According to the synthesis method in example 1, 318mg of CheGDLV was obtained in 31.06% yield. HPLC: t R ＝13.90min.MS(ESI)calcd for C 25 H 44 N 4 O 7 [M-H] - :511.3；found:m/z 511.3. 1 H NMR(300MHz,DMSO-d 6 )δ8.75(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)
151.5mg of CheGDFV was obtained according to the synthesis procedure as in example 1 with a yield of 13.74%. HPLC: t R ＝14.15min.MS(ESI)calcd for C 28 H 42 N 4 O 7 [M+H] + :547.3；found:m/z 547.3. 1 H NMR(300MHz,DMSO-d 6 )δ12.62(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 procedure in example 1. HPLC: t R ＝14.10min.MS(ESI)calcd for C 25 H 44 N 4 O 7 [M+H] + :586.3；found:m/z 586.3. 1 H NMR(300MHz,DMSO-d 6 )δ10.78(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 (CheAD (OMe) AV)
Che-AD (OMe) AV 88mg was obtained in 16.7% yield according to the synthesis method of example 1. HPLC: t R ＝20min. 1 HNMR(400MHz,)δ8.88(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 fluorescence polarization method for testing nNOS-Capon method
1.GST-nNOS 1-133 Induction expression and purification of proteins
Will contain pGEX4T-1-GST-nNOS 1-133 The E.Coli BL21 (DE 3) broth was inoculated into 5mL of LB medium containing 100. Mu.g/mL ampicillin and incubated overnight on a shaker at 37℃and 180 r/min. The next day, transfer to 300mL LB medium containing 100. Mu.g/mL ampicillin, culture for 2.5h, add lactose to induce it to a final concentration of 2.5g/mL, and continue culture for 6h. Centrifugation at 3000g at 4℃for 30min, collection of pellet, re-suspension in cold (concentration: 10mM, 7.4), addition of lysozyme, shaking and mixing, and placing in a-20℃refrigerator overnight. Secondary timesThe bacterial liquid is put on a shaking table, melted again at 37 ℃ and 180r/min, and then DNA enzyme is added to digest the genome DNA. Centrifuge at 15000rpm for 15min at 4℃and collect the supernatant, which was filtered through a 0.22 μm microporous filter. The collected supernatant was applied to a GST affinity column, eluted with reduced glutathione (0.154 g/50mL PBS), and purified to give GST-nNOS 1-133 Protein concentration was performed by ultrafiltration concentration in a 30kDa ultrafiltration tube and protein concentration was determined by BCA kit.
2. Identification of GST-nNOS by Coomassie Brilliant blue staining method 1-133 Proteins
The purified and concentrated protein was subjected to SDS-PAGE, followed by staining overnight with coomassie brilliant blue staining solution, eluting the protein with decolorizing solution the next day, and white light photographing, and the result is shown in FIG. 3, expressed GST-nNOS 1-133 Correct and high purity.
Fitting fluorescent molecular probes 5-FAM-KV-14 and GST-nNOS by isothermal titration calorimetry (isothermal titration calorimetry, ITC) 1-133 And in the binding process of the protein, the binding is verified to be a typical single-point binding model, and the screening requirement of a fluorescence polarization method is met. As shown in FIG. 4, the ratio of probe to protein concentration at saturation is about 10:1, which provides a reference for subsequent fluorescent molecular probe and protein concentration selections.
3. Determination of optimal fluorescent molecular probe concentration
The effect of different concentrations of fluorescent molecular probe 5-FAM-KV-14 on FP values was determined. 5-FAM-KV-14 standard solution (500 mu M) and 1xHepes are respectively added into a 96-well plate according to calculated volumes, so that the final concentration of the fluorescent molecular probe 5-FAM-KV-14 in each well is 100 mu L,3 parallel wells are arranged in each well, the FP value is measured by a multifunctional enzyme-labeled instrument at 37 ℃, and the total volume of each well is plotted through statistical software Graphpad Prism 7.
As shown in FIG. 5, the FP value decreased with increasing concentration of 5-FAM-KV-14 in the probe well containing only free fluorescent molecules. At 50nM of 5-FAM-KV-14, the FP value detected at this time was 57.25mP and became stable as the probe concentration increased. Thus, the final 5-FAM-KV-14 concentration was 50nM.
4. Optimum GST-nNOS 1-133 Determination of protein concentration
Under the condition that the optimal fluorescent molecular probe concentration (50 nM) is determined, GST-nNOS with different concentrations are determined 1-133 Effect of protein on FP values. 5-FAM-KV-14 standard solution (500. Mu.M) and 1xHepes were added to the 96-well plates in the calculated volumes to give a final concentration of 5-FAM-KV-14 in the wells of 50nM. Followed by addition of calculated volumes of GST-nNOS 1-133 The final concentration of the protein stock (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 parallel wells were set up for each concentration, and FP values were measured with a multifunctional microplate reader at 37℃and plotted by statistical software Graphpad Prism 7.
As shown in FIG. 6, with GST-nNOS 1-133 The FP values tended to increase with increasing protein concentration. The concentration corresponding to 0.8 times of the maximum FP value is the optimal concentration of the protein. As can be seen from the graph, the FP value change interval is most pronounced at a protein concentration of 1. Mu.M, and the protein amount is also relatively low. Thus, the GST-nNOS finally selected 1-133 The protein concentration was 1. Mu.M.
5. Determination of the optimal incubation time
Selecting 5-FAM-KV-14 and GST-nNOS 1-133 The optimal incubation time of the protein can make the detected FP value stronger and more stable, is beneficial to reducing errors caused by environmental factors and improves the accuracy of the screening method.
5-FAM-KV-14 (final concentration: 50 nM), GST-nNOS at various concentrations were added to 96-well plates 1-133 The total volume of each hole of the protein and the 1xHepes is controlled to be 125 mu L, and the protein and the 1xHepes are respectively incubated for 1h, 2h, 4h, 8h and 24h at 37 ℃, and three parallel holes are respectively arranged at different reaction times. FP values were determined separately after incubation for various times at 37 ℃ using a multifunctional microplate reader and plotted by statistical software Graphpad Prism 7.
As shown in FIG. 7, GST-nNOS 1-133 After 1h, 2h, 4h, 8h and 24h incubation, the FP value change is basically stable, so that the GST-nNOS which is finally selected 1-133 The protein incubation time was 1h.
Effect of DMSO on FP values
Because some compounds have poor solubility, small amounts of DMSO need to be added, and therefore different levels of DMSO need to be assayed for FP worth of effect.
5-FAM-KV-14 (final concentration: 50 nM), GST-nNOS were added to 96-well plates 1-133 Protein (final concentration: 1. Mu.M), 1xHepes and various volumes of DMSO were controlled to a total volume of 125. Mu.L per well with DMSO content of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 8%, 10%, 12% of the total volume, respectively. FP values were determined separately after incubation for various times at 37 ℃ using a multifunctional microplate reader and plotted by statistical software Graphpad Prism 7.
As shown in FIG. 8, the DMSO content was up to 6%, and the FP value was not substantially affected, and was lower when the value was 6% or more. Therefore, the DMSO content was chosen to be up to 6%.
7. Reliability by adopting Z factor and signal-to-noise ratio evaluation method
After the conditions such as concentration and incubation time are determined, 3 parallel wells are set on a 96-well plate, and GST-nNOS is respectively measured 1-133 The protein (1. Mu.M) was reacted with 5-FAM-KV-14 (50 nM) for 1h of fluorescence polarization (FP, mP). The Z' value and signal to noise ratio (S/N) of the fluorescence polarization system were calculated according to the following formula, and the reliability of the system was evaluated.
Z’＝1–(3σ binding +3σ free )/|μ binding -μ free |；S/N＝(μ binding -μ free )/σ free (wherein sigma is standard deviation, mu is mean value, binding: 5-FAM-KV-14+GST-nNOS) 1-133 FP value of (free): FP value of free 5-FAM-KV-14)
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 fluorescence polarization screening method established by the experiment has the advantages of sensitivity, reliability and the like, and can be better used for screening nNOS-CAPON channel small molecule inhibitors.
8. Screening of Compounds
A calculated volume of a fluorescent probe molecule 5-FAM-KV-14 solution (final concentration: 50 nM), GST-nNOS was added to a 96-well plate 1-133 Protein solution (final concentration: 1. Mu.M) and small molecule inhibitor (final concentrationConcentration 1 mM), 3 replicates were set for each concentration, total competitive inhibition volume was controlled at 125. Mu.L, FP values were determined after incubation for 1h at 37℃after mixing, and the primary screening results are shown in FIG. 9.
Further accurate test results are shown in fig. 10, and the synthesized target compounds are screened through competitive fluorescence polarization experiments to obtain 5 compounds with binding activity, which 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 institutional animal care and use committee of the university of south Beijing medical science. All efforts are made to reduce the number of mice used and their pain. In this study we used Sprague-Dawley rats (9-10 weeks old, 250-270 g) purchased from the university animal center of south Tong. The indoor temperature 22+ -2deg.C and illumination (12 hr light and shade period) were maintained, and food and water were obtained at random. Animals were randomly assigned to experimental groups by computer. The experimenter marks all animals according to a randomization table prior to distribution. The experiments were performed by researchers not aware of the animal attribution experimental group.
Experimental protocols refer to the transient cerebral ischemia model of Zhou L et al. Briefly, chloral hydrate (350 mg/kg, ip) anesthetized SD rats, a 4/0 surgical nylon monofilament rounded tip was introduced into the left internal carotid artery through the external carotid stump, and advanced 20-21mm at the carotid bifurcation until a slight resistance was felt. At this time, the endoluminal filaments block all blood sources of the middle cerebral artery, the internal carotid artery, the anterior cerebral artery, and the posterior cerebral artery. Throughout the process, body temperature was maintained at 37±0.5 ℃. The tether was left in place for 120min, and then the reperfusion was removed. Immediately after removal of the tether, administration was via the tail vein. In the sham operated animals, the occlusion filaments were inserted only 7mm above the carotid bifurcation.
Evaluation results
Infarct volume measurements were performed 24 hours after MACO. The brains were removed rapidly and frozen at-20℃for 5min. Making 1-2 mm coronal slice, immersing slice in 2% TTC at 37 deg.C for 4 hr. The staining results are shown in fig. 11 and 12, and the infarct volume is expressed as a 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 (4)

1. A polypeptide having nNOS-Capon uncoupling activity, characterized by the structural formula Che-Ala-Asp (OMe) -Ala-Val, wherein Che = N-Cyclohexylethyl.
2. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof in the manufacture of a neuroprotective medicament.
3. Use of the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for stroke, anxiety or depression.
4. A neuroprotective agent comprising the polypeptide of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
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US6103872A (en) * 1998-01-22 2000-08-15 The Johns Hopkins University CAPON: a protein associated with neuronal nitric oxide synthase
CN103193669A (en) * 2013-02-27 2013-07-10 南京医科大学 nNOS-Capon uncoupling compound, preparation method and application thereof
CN106928322A (en) * 2017-03-13 2017-07-07 河北科技大学 A kind of fused polypeptide and its application with treating cerebral ischemia

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US6103872A (en) * 1998-01-22 2000-08-15 The Johns Hopkins University CAPON: a protein associated with neuronal nitric oxide synthase
CN103193669A (en) * 2013-02-27 2013-07-10 南京医科大学 nNOS-Capon uncoupling compound, preparation method and application thereof
CN106928322A (en) * 2017-03-13 2017-07-07 河北科技大学 A kind of fused polypeptide and its application with treating cerebral ischemia

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