A polypeptide NR6 with dual regulation function of DBH and DDC enzyme activities and its application

Abstract

The present invention provides a polypeptide NR6 having a dual function of regulating the activity of DBH and DDC enzymes and its application, belonging to the technical field of functional polypeptides. The present invention provides a polypeptide NR6 for the research of interfering small peptides that can target lipopolysaccharide binding protein and dopamine β-hydroxylase and aromatic L-amino acid decarboxylase, which has not yet been seen, and has the function of promoting the activity of dopamine β-hydroxylase and aromatic L-amino acid decarboxylase, thereby promoting the release of dopamine, norepinephrine, epinephrine, serotonin and melatonin in the body, and has anti-inflammatory, anti-depressant, memory recovery, sleep improvement, mental recovery and other functions. In addition, NR6 and its series of small peptides can penetrate the blood-brain barrier and have the advantages of small molecular weight and high stability, which are conducive to commercial application.

Description

Technical Field

[0001] The invention belongs to the technical field of functional polypeptides, and specifically relates to a polypeptide NR6 having dual functions of regulating DBH and DDC enzyme activities and an application thereof. Background Art

[0002] Among the enzymes in the catecholamine pathway, dopamine β-hydroxylase (DBH) is an important neurotransmitter synthase that catalyzes dopamine (DA) to form norepinephrine (NE). Since its discovery in 1950, it has been widely studied. (1,2) The catecholamine pathway is considered the sole source of NE and epinephrine (EPI), which is thought to regulate many processes such as mood, general arousal, attention, sexual behavior, stress, immunity, learning, and memory. (3) NE acts as a neurotransmitter in both the central and peripheral nervous systems and is a precursor for adrenaline synthesis in the brain and adrenal medulla. Alterations in noradrenergic signaling have been associated with central and peripheral nervous system pathologies. (1,2) Therefore, DBH protein in the peripheral circulation can be assessed as a marker of norepinephrine-related function in a variety of different diseases and disorders. In many of these diseases, the activity of DBH protein contributes to disease characterization. Decreased DBH activity directly leads to lower NE and epinephrine levels, and the associated diseases eventually develop. (4-6) .

[0003] Aromatic L-amino acid decarboxylase (DDC) was first identified from mammalian kidney extracts in 1938. In addition to its ability to convert L-DOPA (L-DOPA) into dopamine (DA) and participate in the regulation of catecholamines, it also has the ability to convert L-5-hydroxytryptophan (5-HTP) into serotonin (5-hydroxytryptamine, 5-HT). (7,8) The decrease in DDC content or activity will lead to a decrease in the content of neurotransmitters DA, 5-HT and sleep-regulating hormone melatonin, leading to depression, reduced memory, attention deficit, sleep disorders, etc. (7,8) Therefore, means of regulating the activity of DBH and AADC would benefit the treatment of diseases related to their products, such as hypertension (ranked as the world's largest disease burden), congestive heart failure, Alzheimer's disease and drug addiction, as well as Parkinson's disease, Huntington's disease, Tourette syndrome, depression and attention deficit hyperactivity disorder.

[0004] A literature search revealed no reports on polypeptides or proteins that can simultaneously regulate the activities of dopamine β-hydroxylase and aromatic L-amino acid decarboxylase.

[0005] References

[0006] 1.D.N.Catelas,M.P.Serrao,P.Soares-Da-Silva,Effects of nepicastat upondopamine-beta-hydroxylase activity and dopamine and norepinephrine levels inthe rat left ventricle,kidney,and adrenal gland.Clin Exp Hypertens42,118-125(2020).

[0007] 2.T.V.Vendelboe et al.,The crystal structure of human dopamine beta-hydroxylase at2.9A resolution.Sci Adv2,e1500980(2016).

[0008] 3.J.J.Schildkraut,S.S.Kety,Biogenic Amines and Emotion.Science156,21-+(1967).

[0009] 4.M.K.Sapru,B.S.Rao,S.M.Channabasavanna,Serum dopamine-beta-hydroxylase activity in clinical subtypes of depression.Acta PsychiatrScand80,474-478(1989).

[0010] 5.B.S.Meyers et al.,Decreased dopamine beta-hydroxylase activity inunipolar geriatric delusional depression.Biol Psychiatry45,448-452(1999).

[0011] 6.JFCubells et al., Genotype-controlled analysis of plasma dopaminebeta-hydroxylase activity in psychotic unipolar major depression. BiolPsychiatry51,358-364(2002).

[0012] 7. WLHwu, NCLee, YHChien, S.Muramatsu, H.Ichinose, AADC deficiency: occurring in humans, modeled in rodents. Adv Pharmacol68, 273-284 (2013).

[0013] 8.MDBerry,AVJuorio,XMLi,AABoulton,Aromatic L-amino aciddecarboxylase:a neglected and misunderstood enzyme.Neurochem Res21,1075-1087(1996). Summary of the invention

[0014] In view of this, the object of the present invention is to provide a polypeptide NR6 having the function of dually regulating the activities of DBH and DDC enzymes.

[0015] The present invention provides a polypeptide NR6, and the amino acid sequence of the polypeptide NR6 is shown in SEQ ID NO:1.

[0016] The invention provides an enzyme activity promoter for dopamine beta-hydroxylase and aromatic L-amino acid decarboxylase, comprising the polypeptide NR6 and auxiliary materials.

[0017] The present invention provides the use of the polypeptide NR6 in preparing a product for improving the enzymatic activity of dopamine β-hydroxylase and / or aromatic L-amino acid decarboxylase.

[0018] Preferably, the enzymatic activity of dopamine β-hydroxylase and / or aromatic L-amino acid decarboxylase is increased by interfering with the binding of lipopolysaccharide binding protein LBP with dopamine β-hydroxylase and / or aromatic L-amino acid decarboxylase through polypeptide NR6, thereby increasing the enzymatic activity of dopamine β-hydroxylase and / or aromatic L-amino acid decarboxylase.

[0019] The present invention provides the use of the polypeptide NR6 or the enzyme activity promoter in preparing a drug for increasing blood pressure.

[0020] The present invention provides the use of the polypeptide NR6 or the enzyme activity promoter in the preparation of anti-inflammatory drugs.

[0021] The present invention provides the use of the polypeptide NR6 or the enzyme activity promoter in the preparation of medicines for preventing and treating mental illness.

[0022] Preferably, the psychiatric disorder comprises at least one of the following: depression, Alzheimer's disease, Parkinson's disease, schizophrenia, attention deficit hyperactivity disorder and sleep disorder.

[0023] The present invention provides application of the polypeptide NR6 or the enzyme activity promoter in preparing a medicine for depression.

[0024] The present invention provides a polypeptide NR6, the amino acid sequence of which is shown in SEQ ID NO: 1. The present invention is the first to discover an interfering small peptide that can target LBP-DDC / DBH, which has the function of promoting the activity of DDC and DBH, thereby promoting the release of dopamine (DA), norepinephrine (NE), 5-hydroxytryptamine (5-HT) and metallothionein (MT), and has the potential to be a treatment for hypertension, anti-inflammation, and mental illness (depression, Alzheimer's disease, Parkinson's disease, schizophrenia, attention deficit hyperactivity disorder and sleep disorders). BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of SPR analysis of the interference of NR6 on the binding of LBP to DDC and the binding of LBP to DBH;

[0026] Figure 2 The results of RP-HPLC analysis of the effect of NR6 on the inhibitory activity of LBP against DDC;

[0027] Figure 3 The results of enzyme kinetic analysis of the effect of NR6 peptide on DBH activity;

[0028] Figure 4 The results of NR6's effect on blood pressure in mice;

[0029] Figure 5 The results of pharmacokinetic analysis of NR6 in mouse brain;

[0030] Figure 6 The results show the effect of NR6 on LPS-induced decrease of DBH activity and NE content in mice;

[0031] Figure 7 The results show the effect of NR6 on the depressive behavior of LPS-induced mice;

[0032] Figure 8The results of the eight-arm maze analysis of the effect of NR6 on the memory of LPS-induced mice;

[0033] Fig. 9 Immunofluorescence analysis of the effect of NR6 on LPS-induced neurogenesis and microglia in the mouse brain;

[0034] Fig.10 This is the result of enzyme-linked immunosorbent assay of the effect of NR6 on LPS-induced brain inflammation in mice. DETAILED DESCRIPTION

[0035] The present invention provides a polypeptide NR6, the amino acid sequence of the polypeptide NR6 is shown in SEQ ID NO: 1 (NVQYMR, wherein the amino acids are L-type amino acids).

[0036] In the present invention, the preparation method of the polypeptide NR6 is preferably a polypeptide synthesis method known in the art. In the embodiment of the present invention, the polypeptide NR6 is synthesized by commissioning Gill Biochemical Company.

[0037] In the present invention, in vitro and in vivo experiments were conducted on the biological functions of polypeptide NR6. In vitro, surface plasmon resonance (SPR) experiments were used to determine that NR6 had an interfering effect on the binding of LBP-DDC / DBH; reverse phase high performance liquid chromatography (RP-HPLC) substrate identification method was used to determine that NR6 had a promoting effect on DDC activity; and a chromogenic substrate method was used to determine that polypeptide NR6 also had a promoting effect on DBH activity. In vivo, it was determined that polypeptide NR6 had an effect of increasing blood pressure on mice; it was determined that polypeptide NR6 had the ability to penetrate the mouse blood-brain barrier, and the pharmacokinetic analysis results in the mouse brain showed that the half-life y of polypeptide NR6 in the mouse brain was about 4 hours, and it had a long drug effect. This shows that polypeptide NR6 has the prerequisite for preparing drugs for treating cranial nerve diseases. At the same time, the peptide NR6 has the function of restoring the depressive-like phenotype (behavioral analysis such as sleep, memory, emotion and histopathological analysis) of stress-induced depression model mice, and has the pharmacological effect of preparing drugs for treating depression; in addition, the peptide NR6 also has an inhibitory function on inflammation in the mouse inflammation model, which indicates that the peptide NR6 has the pharmacological effect of inhibiting inflammation.

[0038] Based on the biological effect of the polypeptide NR6 in simultaneously improving the enzymatic activities of dopamine β-hydroxylase and aromatic L-amino acid decarboxylase, the present invention provides an enzyme activity promoter for dopamine β-hydroxylase and aromatic L-amino acid decarboxylase, comprising the polypeptide NR6 and auxiliary materials.

[0039] In the present invention, there is no special limitation on the preparation method of the enzyme activity promoter, and any preparation method of the enzyme activity promoter known in the art can be used. There is no special limitation on the type of the auxiliary materials, and any auxiliary materials of the enzyme activity promoter known in the art can be used.

[0040] The present invention also provides the use of the polypeptide NR6 in preparing a product for improving the enzymatic activity of dopamine β-hydroxylase and / or aromatic L-amino acid decarboxylase.

[0041] In the present invention, the improvement of dopamine β-hydroxylase and / or aromatic L-amino acid decarboxylase enzyme activity is preferably achieved by interfering with the binding of lipopolysaccharide binding protein LBP with dopamine β-hydroxylase and / or aromatic L-amino acid decarboxylase through polypeptide NR6, thereby improving the dopamine β-hydroxylase and / or aromatic L-amino acid decarboxylase enzyme activity.

[0042] The present invention provides the use of the polypeptide NR6 or the enzyme activity promoter in preparing a drug for increasing blood pressure.

[0043] In the present invention, the results of mouse experiments show that the NR6 has the effect of increasing blood pressure when the concentration is 0.5-1 mg / kg, and the duration of blood pressure increase is preferably 7-9 minutes.

[0044] The present invention provides the use of the polypeptide NR6 or the enzyme activity promoter in the preparation of anti-inflammatory drugs.

[0045] In the present invention, NR6 inhibited the decrease of DBH activity and NE content in the brain of mice induced by inflammatory stress, while the positive control idaoxan had no effect in a short period of time. The polypeptide NR6 also reversed the aggravation of neuroinflammation in the DG region induced by LPS, and inhibited the production of TNF-α and IL-6 in the brain.

[0046] In view of the fact that the polypeptide NR6 has the function of promoting the enzymatic activity of dopamine β-hydroxylase and / or aromatic L-amino acid decarboxylase, and dopamine β-hydroxylase has the function of catalyzing the formation of NE, and aromatic L-amino acid decarboxylase has the function of forming DA, 5-HT and promoting the production of downstream MT, the polypeptide NR6 can promote the release of DA, NE, 5-HT and MT, and these components play an important role in mental illness. Therefore, the present invention provides the use of the polypeptide NR6 or the enzyme activity promoter in the preparation of drugs for preventing and treating mental illness.

[0047] In the present invention, the mental illness preferably includes at least one of the following: depression, Alzheimer's disease, Parkinson's disease, schizophrenia, attention deficit hyperactivity disorder and sleep disorder.

[0048] The present invention provides application of the polypeptide NR6 or the enzyme activity promoter in preparing a medicine for depression.

[0049] In the present invention, the depression preferably includes acute stress-induced depression. In an embodiment of the present invention, the acute stress-induced depression mouse model is induced by intravenous injection of 1 mg / kg LPS. In an embodiment of the present invention, the behavioral test results of the grip test (GST), social interaction test (SIT), tail suspension test (TST), forced swimming test (FST) and eight-arm maze (RAM) task show that NR6 has significant antidepressant-like behavioral functions.

[0050] The following is a detailed description of the polypeptide NR6 having dual regulation function of DBH and DDC enzyme activities and its application provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] Surface plasmon resonance detection of NR6 interference with LBP-DDC / DBH binding

[0053] The LBP protein was bound to a CM5 chip (the chip was activated using 0.4 M EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) and 10 mM NHS (N-Hydroxysuccinimide), and blocked with 1 M ethanolamine), and the binding of a mixture of NR6 polypeptide (NVQYMR, SEQ ID NO: 1) with different concentrations (0-10 μM) and DBH or DDC protein was detected using a surface plasmon resonance instrument to obtain the binding response values ​​of the mixture of NR polypeptide with different concentrations (0-10 μM) and DBH or DDC protein with LBP protein.

[0054] like Figure 1 As shown, after the addition of NR6, the binding response value of DDC or DBH and LBP was reduced, indicating that NR6 interfered with the binding of DDC or DBH and LBP.

[0055] Example 2

[0056] RP-HPLC detection of the interference of NR6 peptide on the inhibitory activity of LBP on DDC

[0057] 50 μl of a mixture of 2 μM DDC protein and LBP (20 nM) and / or different concentrations of NR6 (0-5 μM) was incubated with 50 μl 0.7 mM pyridoxal-5-phosphate (containing 39 mM dithiothreitol and 0.167 mM NaEDTA) at 37 ° C for 2 hours. Subsequently, 100 μl of L-DOPA (2 mM) was added and incubated at 37 ° C for 2 hours. The reaction solution was ultrafiltered to remove the enzyme and other macromolecules, and then placed on ice to terminate the reaction. The sample was analyzed by RP-HPLC using a C18 column. Deionized water was solvent A liquid, acetonitrile was elution buffer B liquid and eluted with a 0.1% gradient, and the absorbance was monitored at a flow rate of 0.5 ml / min at 280 nm. In order to identify the peak of L-DOPA, the effluent of each peak was collected and used for mass spectrometry qualitative analysis. RP-HPLC analyzed the corresponding L-DOPA standard curve to calculate the DDC enzyme activity of the sample.

[0058] like Figure 2 As shown, LBP inhibited the activity of DDC, and after NR6 treatment, the activity of DDC was increased, indicating that NR6 interfered with the inhibition of DDC activity by LBP.

[0059] Example 3

[0060] Detection of the promoting effect of NR6 peptide on DBH activity by chromogenic substrate method

[0061] 10μl 100μg / ml DBH was mixed with 10μl NR6 peptide and / or 10μl LBP (20nM) of different concentrations (0-5μM), and 100μl N,N-dimethyl-1,4-phenylenediamine (DMPD, 1.4mM, diluted with 0.01M PBS, pH7.2) was added to the mixture. DBH has the function of promoting the oxidation and color development of the substrate DMPD, so the DBH activity analysis was performed by continuous colorimetric determination. The absorbance at 515nm was monitored, and the kinetic curve was recorded for 30 minutes using an enzyme reader. Due to the automatic oxidation of DMPD, a corresponding substrate control was set in each test.

[0062] like Figure 3 As shown, NR6 interferes with LBP's inhibition of DBH protein activity.

[0063] Example 4

[0064] Effect of NR6 peptide on blood pressure in mice

[0065] The same mouse was dosed with different concentrations (0.25-2 mg / kg) of NR6 polypeptide drugs and the concentration was changed after an interval of 36 hours for retesting. Blood pressure was monitored once before administration and blood pressure was tested using a blood pressure and heart rate monitor immediately after administration.

[0066] like Figure 4 As shown, NR6 polypeptide has the effect of increasing blood pressure at concentrations of 0.5 mg / kg and 1 mg / kg, and the duration is about 8 minutes.

[0067] Example 5

[0068] Pharmacokinetic analysis of NR6 peptide

[0069] To analyze the metabolism of NR6 peptide in mouse brain, brain homogenates were collected at different time points after intravenous injection of 4 mg / kg NR6 peptide and delivered to the Shared Technology and Facilities Center of Kunming Institute of Zoology, Chinese Academy of Sciences for liquid chromatography-mass spectrometry (LC / MS) analysis by technician Zeng Lin.

[0070] like Figure 5 As shown, the NR6 polypeptide showed a good ability to penetrate the blood-brain barrier, with a half-life of about 4 hours in the mouse brain.

[0071] Example 6

[0072] Effects of NR6 on depression-like phenotypes and inflammation in LPS-induced depression model mice

[0073] In order to determine the efficacy of NR6 in the treatment of acute stress-induced depression, 8-week-old male C57BL / 6J mice were intravenously injected with 1 mg / kg LPS for 24 hours, and then a social interaction test was performed. The specific method consisted of two 2.5-minute stages, in which the mice were placed in an open area (45×45×45 cm) with a visible and ventilated circular isolation cage (10 cm in diameter). In the first stage, the isolation cage was empty (no target), and 2.5 minutes were recorded. In the second stage, 7-month-old CD1 mice (unfamiliar) were placed in the isolation cage (target) and recorded for 2.5 minutes. Between these two stages, the experimental mice were returned to the mouse cage for 30 seconds. The time spent in the social area by the test mice was analyzed using the SMART video tracking system v3.0. Compared with the PBS-injected group, the LPS injection 24 hours later showed a lower social interaction time in the social interaction test, and the obvious depressive-like phenotype showed that the present invention successfully constructed an acute stress-induced depression model mouse.

[0074] The same molar concentration of NR6 peptide (4 mg / kg) and idazoxan (1 mg / kg) were intravenously injected into mice with acute stress-induced depression. After 30 minutes, part of the mouse brain homogenate was used to determine DBH activity, NE and LBP content, and the other part of the mice was used for grasping test (GST), social interaction test (SIT), tail suspension test (TST), sucrose preference test (PST) or forced swimming test (FST).

[0075] In addition, mice were subjected to an eight-arm maze (RAM) task after daily intravenous injection of NR6 peptide (4 mg / kg) or combined intravenous injection of LPS (0.1 mg / kg) and intraperitoneal injection of BrdU (50 mg / kg). After the RAM task, a portion of the brain was used for immunofluorescence analysis of neurogenesis and microglial cell number in the hippocampus (DG), and another portion of the brain homogenate was used for enzyme-linked immunosorbent assay (ELISA) of IL-6 and TNF-α content.

[0076] TST: The tails of mice were glued to a stand 30 cm above the ground and video recorded during the 6-min test (1-min acclimatization time and 5-min recording). The time of immobility was automatically determined using the Intelligent Video Tracking System v3.0.

[0077] FST: Mice were placed in a round transparent beaker with a diameter of 11.2 cm and a water depth of 11 cm for 6 min at 25°C. The immobility time was automatically determined using the Intelligent Video Tracking System v3.0.

[0078] SIT: It consisted of two 2.5-min phases in which mice were placed in an open area (45 × 45 × 45 cm) with a visible and ventilated circular isolation cage (10 cm diameter). In the first phase, the isolation cage was empty (no target) and recorded for 2.5 min. In the second phase, a 7-month-old CD1 mouse (unfamiliar) was placed in the isolation cage (target) and recorded for 2.5 min. Between these two phases, the experimental mouse was returned to the mouse cage for 30 s. The time the test mice spent in the social area was analyzed using the SMART video tracking system v3.0.

[0079] SPT: Mice were acclimated to a 2-bottle water delivery system (one bottle containing 1% sucrose and one bottle containing water) for 3 days, and then sucrose preference was assessed on two consecutive days. Sucrose preference was calculated by dividing the amount of sugar water consumed by the total volume consumed (water + sucrose).

[0080] GST: GST of mice was tested using a handgrip dynamometer (DS2-50N, Sansbio, China) according to the manufacturer's instructions.

[0081] RAM: used to detect the spatial reference memory of mice. In order to adapt to the maze and baits (pre-training), mice were allowed to move freely in the RAM twice a day for 2 days, and food baits were distributed at the ends of all arms. The training trial started after pre-training. During the training trial, food baits were placed in the same 2 arms. After the mouse left the entrance arm and entered the center of the arm, the closed entry arm was used as the spatial reference of the mouse. Each mouse was tested 2 times a day for 5 days. The test ended until the mouse received two rewards or 5 minutes later. Entering an arm where no bait was placed was considered a reference memory error, and the number of entries into the wrong arm was recorded.

[0082] like Figure 6 As shown, 30 minutes after administration, 4 mg / kg NR6 peptide inhibited the decrease of DBH activity and NE content in the mouse brain induced by inflammatory stress, while the positive control idaoxan had no effect in a short period of time. Figure 7 and 8 The behavioral test results of GST, SIT, TST, FST and RAM shown in the figure showed that NR6 had significant antidepressant-like behavioral functions.

[0083] In addition, NR6 reversed the LPS-induced decrease in neurogenesis and increase in neuroinflammation (microglia-specific Iba1-positive cells) in the DG region (see Fig. 9 ), and inhibited the production of TNF-α and IL-6 in the brain (see Fig.10 ).

[0084] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide NR6, characterized in that: The amino acid sequence of the polypeptide NR6 is shown in SEQ ID NO:

1.

2. A dopamine β-hydroxylase and aromatic L-amino acid decarboxylase enzyme activity promoter, characterized in that: The invention comprises the polypeptide NR6 according to claim 1 and auxiliary materials.

3. Use of the polypeptide NR6 according to claim 1 or the enzyme activity promoter according to claim 2 in the preparation of a drug for increasing blood pressure.

4. Use of the polypeptide NR6 according to claim 1 or the enzyme activity promoter according to claim 2 in the preparation of a drug for treating depression.

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