Polypeptide with antihypertensive activity and application

Abstract

The invention discloses a polypeptide with antihypertensive activity. The N and C terminals of the polypeptide are aromatic amino acid and proline. The polypeptide contains 3-5 amino acid sequences. The polypeptide is any one of five polypeptides of which amino acids are respectively FGPW, PFPRP, PFA, NPF and FPPP. The invention also provides application of the polypeptide in antihypertensive drugs, five polypeptides are designed according to the principle that N and C terminals are aromatic amino acids and proline, the simulated peptide fragment interacts with ACE, and the polypeptide has a good antihypertensive effect, is non-toxic and can be used for developing antihypertensive drugs.

Description

Technical Field

[0001] The present invention relates to the field of polypeptides for lowering blood pressure, and more specifically, to a polypeptide having blood pressure lowering activity and its use. Background Art

[0002] Hypertension is an important factor leading to diseases such as stroke, coronary heart disease, and heart failure. As a chronic disease, most patients with hypertension need to take antihypertensive drugs for a long time or for life. According to the "Key Points of Clinical Medication Instructions of the People's Republic of China", antihypertensive drugs are divided into seven categories, but each type of drug has different side effects. The blood pressure compliance rate of hypertensive patients in my country is not high, which is related to factors such as poor efficacy of monotherapy, drug tolerability and poor patient compliance. Therefore, the development of new antihypertensive drugs with new targets is of great clinical significance to improve the blood pressure compliance rate.

[0003] With the development and maturity of peptide synthesis technology, peptide drugs have become one of the hot spots in drug research and development. Due to their wide indications, high safety and significant efficacy, they have been widely used in the prevention, diagnosis and treatment of diseases such as tumors, cardiovascular and cerebrovascular diseases, hepatitis, diabetes, AIDS, etc., and have broad development prospects. Summary of the invention

[0004] In order to achieve these purposes and other advantages according to the present invention, on the one hand, a preferred embodiment of the present invention provides a polypeptide having blood pressure lowering activity, wherein the N- and C-termini are aromatic amino acids and proline.

[0005] According to a preferred embodiment of the present invention, the polypeptide comprises 3-5 amino acid sequences.

[0006] According to a preferred embodiment of the present invention, the polypeptide is any one of the five polypeptides whose amino acids are FGPW, PFPRP, PFA, NPF, and FPPP respectively.

[0007] On the other hand, another preferred embodiment of the present invention provides a use of the polypeptide in the preparation of a drug for treating hypotensive disease.

[0008] The present invention has at least the following beneficial effects: the present invention designs 5 polypeptides based on the principle that the N and C termini are aromatic amino acids and proline, simulates the interaction between peptide segments and ACE, and proves through in vitro experiments and animal experiments that the 5 polypeptides all have antihypertensive effects, and the polypeptide with the highest antihypertensive activity is PFPRP. The polypeptide of the present invention has a good antihypertensive effect, is non-toxic, and can be used for the development of antihypertensive drugs.

[0009] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The docking site and details of FGPW and ACE.

[0011] Figure 2 The docking site and details of PFPRP and ACE.

[0012] Figure 3 The docking site and details of PFA and ACE.

[0013] Figure 4 The docking site and details of NPF and ACE.

[0014] Figure 5 The docking site and details of FPPP and ACE. DETAILED DESCRIPTION

[0015] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0016] The following description is used to disclose the present invention so that those skilled in the art can implement the present invention. The preferred embodiments described below are only examples, and those skilled in the art can think of other obvious variations. The basic principles of the present invention defined in the following description can be applied to other embodiments, variations, improvements, equivalents, and other technical solutions that do not deviate from the spirit and scope of the present invention.

[0017] Experiment 1: Interaction between simulated peptide and angiotensin converting enzyme (ACE)

[0018] According to the principle of aromatic amino acids and proline at the N and C termini, five peptides were designed with 3-5 amino acids, and the number of designed peptides was 5, namely FGPW, PFPRP, PFA, NPF, and FPPP.

[0019] The crystal structure of angiotensin converting enzyme (ACE) (PDB number: 108A) was obtained from the RCSB database (http: / / www.rcsb.org / ), and semi-flexible molecular docking was performed on the five peptides using AutoDock 4.2.6 software. Table 1 shows the active peptide sequences and molecular docking scoring results.

[0020] Table 1 Peptide sequences and molecular docking scoring results

[0021]

[0022]

[0023] The five designed peptides were semi-flexibly docked with angiotensin-converting enzyme (ACE). All five peptides were able to form stable conformations with ACE, and the intermolecular forces included hydrogen bonds, van der Waals forces, etc.

[0024] The FGPW peptide was semi-flexibly docked with angiotensin converting enzyme (ACE), and the interaction force between it and the key amino acid residue ARG-501 of ACE was hydrogen bond. The intermolecular interaction force is shown in Figure A.

[0025] PFPRP peptide was semi-flexibly docked with angiotensin converting enzyme (ACE), and the interaction force between it and the key amino acid residues GLU-176, ALA-261, GLN-262, and HIS-263 of ACE was van der Waals force. The intermolecular interaction force is shown in Figure B.

[0026] PFA peptide is semi-flexibly docked with angiotensin converting enzyme (ACE), and its interaction force with the key amino acid residue GLU-176 of ACE is hydrogen bond. The intermolecular interaction force is shown in Figure C.

[0027] The NPF peptide was semi-flexibly docked with angiotensin converting enzyme (ACE), and the interaction force between it and the key amino acid residue GLU-176 of ACE was hydrogen bond. The intermolecular interaction force is shown in Figure D.

[0028] The FPPP peptide was semi-flexibly docked with angiotensin converting enzyme (ACE), and the interaction force between it and the key amino acid residues HIS-263 and ASN-265 of ACE was hydrogen bond. The intermolecular interaction force is shown in Figure E.

[0029] Test 2

[0030] The physicochemical properties of the five peptides FGPW, PFPRP, PFA, NPF, and FPPP were predicted using relevant software. The toxicity and steric hindrance prediction website is ToxinPred (ToxinPred (iiitd.edu.in)), the overall average hydrophilicity prediction website is ExPasy (Expasy-ProtParam), the human intestinal absorption prediction website is admetSAR (admetSAR@LMMD (ecust.edu.cn)), and the isoelectric point prediction website is pepdraw (PepD http: / / www.pepdraw.com / ).

[0031] The five antihypertensive peptides are non-toxic and have little steric hindrance, which is conducive to binding with ACE. The isoelectric points of the four peptides FGPW, PFA, NPF, and FPPP are less than 7 and are acidic. The isoelectric point of PFPRP is 11.56, which is greater than 7 and is alkaline. The total average hydrophilicity of PFA is positive, indicating that the peptide is highly hydrophobic and insoluble in water. The total average hydrophilicity of the four peptides PGPW, PFPRP, NPF, and FPPP is negative, indicating that they are highly hydrophilic and soluble in water. The two peptides PFA and FPPP have high intestinal absorption rates, while the intestinal absorption rates of FGPW, PFPRP, and NPF are poor. The prediction of physicochemical properties is of reference significance for the subsequent modification of peptides and the development of drug dosage forms.

[0032] Table 2 Prediction of physical and chemical properties

[0033]

[0034] Example 3 In vitro blood pressure lowering experiment

[0035] 3.1 A third party company (Suzhou Modif Biotechnology Co., Ltd.) was commissioned to prepare the five antihypertensive peptides screened using the Fmoc solid phase synthesis method, and the purity of the peptides was determined to be greater than 95% through high performance liquid chromatography and mass spectrometry analysis.

[0036] 3.2 Angiotensin-converting enzyme inhibitory activity assay

[0037] The kit was tested by ultraviolet spectrophotometry (Solabo), and the reagents were checked according to the kit instructions, namely reagent 1, reagent 2, reagent 3, and reagent 4, of which reagent 4 was a positive control. The reagent 3 working solution was prepared according to the steps in the instructions, and 50uL of distilled water was added to dissolve it before use. According to the sample volume, the ratio of reagent 3: reagent 1 = 9uL: 1791uL was prepared, and the reagent 3 working solution was obtained before use.

[0038] FGPW, PFPRP, PFA, NPF, and FPPP were respectively prepared into concentration gradients of 0.5mmol / L, 1mmol / L, 2mmol / L, 4mmol / L, 8mmol / L, 12mmol / L, 16mmol / L, and 20mmol / L, wherein FGPW, PFPRP, NPF, and FPPP were dissolved and diluted to the corresponding concentrations with reagent 1 provided in the kit, and PFA was dissolved with 1% ethanol and then diluted to the corresponding concentration with reagent 1. The obtained samples of different concentrations were placed in the assay tubes, and the samples were added and measured according to the steps in Table 3, and the inhibition rate of each concentration sample was calculated according to formula (1).

[0039] Table 3

[0040]

[0041] ACE inhibitor inhibition rate (%) = (ΔA 空白 -ΔA 测定 )÷ΔA 空白 ×100%(1).

[0042] IC 50 Calculation of inhibition value: draw an inhibition curve with sample concentration as the horizontal axis and inhibition rate as the vertical axis to obtain the sample concentration when the inhibition rate is 50%.

[0043] IC of 5 antihypertensive peptides 50 See Table 5 for values.

[0044] Table 4 IC values ​​of five antihypertensive peptides 50 value

[0045]

[0046] From the data in Table 4, we can see that all five peptides have antihypertensive activity in vitro, among which the IC 50 The lowest value was 2.51mmol / L, and the in vitro antihypertensive activity was the highest. 50 The IC value refers to the inhibitor concentration when the ACE inhibition rate reaches 50%. It is a very accurate indicator for evaluating the in vitro inhibitory activity of ACE inhibitory peptides. 50 The smaller the value, the stronger the activity of the ACE inhibitory peptide. According to the ACE inhibitory activity peptides reported in the literature, IC 50 Below 20 μmol / L, it can show a significant antihypertensive effect. It can be seen that the five short peptides provided in the patent of the present invention all show strong in vitro antihypertensive activity, and the in vitro antihypertensive activity of the PFPRP polypeptide is the highest.

[0047] 3.3 Experiment on the antihypertensive effect of antihypertensive peptides in vivo

[0048] A third-party company (Suzhou Modif Biotechnology Co., Ltd.) was commissioned to prepare the five antihypertensive peptides using the Fmoc solid phase synthesis method, and the peptide purity was determined to be greater than 95% through high performance liquid chromatography and mass spectrometry analysis. The peptide group drugs in the animal experiments were all prepared from the synthetic peptide solid powder.

[0049] Spontaneously hypertensive model rats (SHR), 70, male, 10-12 weeks old, weighing 210-230 g; normotensive rats (WKY), 10, male, 10-12 weeks old, weighing 210-230 g. The temperature of the breeding environment was maintained at (25±1)℃, the humidity was maintained at 55%±5%, the light / dark cycle was 12 hours per day, and the lighting time was from 8:00 am to 20:00 pm. Rats could eat standard feed and tap water at will, and the breeding environment was disinfected regularly. Before the experiment, SHR and WKY had a one-week environmental adaptation period. After the adaptation period, the weight and blood pressure of the rats were measured, and they were divided into different groups according to the measurement results to ensure the uniformity of the baseline data of the rats in each group.

[0050] SHR rats were divided into 7 groups, with 10 rats in each group, namely, the positive control group intervened by captopril, the FGPW polypeptide intervention group, the PFPRP polypeptide intervention group, the PFA polypeptide intervention group, the NPF polypeptide intervention group, the FPPP polypeptide intervention group, and the hypertension model control group. WKY rats served as the blank control group with 10 rats.

[0051] Table 5. Table of oral administration dosage for each group

[0052]

[0053]

[0054] The rats were fed with a normal diet and given drugs by gavage once a day according to the dosage gavage table for 8 consecutive weeks. The body weight, food intake and blood pressure of the rats were collected every week during the experiment.

[0055] When measuring the blood pressure and heart rate of rats, in order to ensure the accuracy of the measured data, the rats to be tested need to be placed in a warm box (37°C) in advance and left to stand for about 15 minutes. After the rats are quiet and their blood pressure is stable, the systolic blood pressure, diastolic blood pressure and heart rate of SHR and WKY rats are monitored using the BP-2006A small animal non-invasive blood pressure meter. The rats are fixed in the heating hood, and a blackout cloth is put on the outer layer of the heating hood to prevent the rats from having a stress response that affects the accuracy of the test data. The rats are placed in the heating hood and covered with a blackout cloth. The temperature of the heating hood is set to 37-39°C to ensure that the tail artery of each rat is fully dilated during the measurement. The sphygmomanometer airbag cuff is put on 3 cm from the tail root of the fixed rat, and the "pulse signal sensor" in the airbag cuff is kept directly above the tail artery. When the sensor shows that the pulse wave is in a stable wave shape, blood pressure measurement begins. The system was connected to a computer, and the test results were recorded using BP98AWU software, recording three to five readings with heart rate changes <10 beats / min and blood pressure changes <20 mmHg, and the measurements were repeated three times for each rat to obtain the average value.

[0056] Each group was observed by gavage for 8 weeks. During the experiment, the animals in each group showed normal growth, development, and activity, and were in good mental state, with no signs of poisoning or death.

[0057] Table 6 Changes in body weight of rats in each group

[0058]

[0059]

[0060] Table 6 shows the changes in body weight of rats in each group after 1 week, 4 weeks, and 8 weeks of administration. There was no significant change in body weight among the groups, and the growth and development, activities, and mental state of the animals in each group were normal during the experiment, and there was no poisoning or death. This shows that the 5 antihypertensive peptides screened have no toxic side effects.

[0061] Table 7 Systolic blood pressure (SBP) of rats in each group after 8 weeks of intervention

[0062]

[0063]

[0064] Table 7 shows the weekly systolic blood pressure test values ​​of rats in each group. In the 0th week of intervention, the diastolic blood pressure of the hypertension model group, each polypeptide group and the positive control group was significantly higher than that of the blank control group (P < 0.05), and the hypertension model was established.

[0065] After 8 weeks of intervention, the blood pressure of the peptide group and the positive control group decreased to a certain extent compared with the hypertension model group. The blood pressure of the FGPW peptide group, PFPRP peptide group and positive control group decreased significantly compared with the hypertension model group (P < 0.05), and the PFPRP peptide group had the largest decrease, which was equivalent to the positive control group.

[0066] Table 8 Diastolic blood pressure (DBP) of rats in each group after 8 weeks of intervention

[0067]

[0068]

[0069] Table 8 shows the weekly diastolic blood pressure test values ​​of rats in each group. In the 0th week of intervention, the diastolic blood pressure of the hypertension model group, the polypeptide groups and the positive control group was significantly higher than that of the blank control group (P < 0.05), and the hypertension model was established.

[0070] At the 8th week of intervention, the diastolic blood pressure of the peptide groups and the positive drug group decreased compared with the hypertension model control group. The diastolic blood pressure of the FGPW peptide and PFPRP peptide groups was significantly lower than that of the hypertension model group (P < 0.05), and the PFPRP peptide group had the largest decrease, and the decrease rate was equivalent to that of the positive control group.

[0071] Table 9 Heart rate (HR) of rats in each group after 8 weeks of intervention

[0072]

[0073]

[0074] Table 9 shows the heart rate detection values ​​of rats in each group after 8 weeks of intervention. The values ​​of the hypertension model control group were significantly higher than those of the blank control group (P < 0.05), and the values ​​of the polypeptide groups and the positive control group were significantly lower than those of the hypertension model control group (P < 0.05), and the heart rate decreased significantly.

[0075] Combined with the results of ACE inhibitor half-inhibition rate test and the results of antihypertensive effect experiment of antihypertensive peptides in vivo, the 5 screened antihypertensive peptides all have antihypertensive effects. The antihypertensive effects of FGPW and PFPRP peptides are significantly better than those of the other three peptides, and the efficacy results of the PFPRP peptide at a dose of 20 mg / kg are equivalent to the efficacy of the positive drug (captopril) at a dose of 10 mg / kg.

[0076] The present invention designs 5 antihypertensive peptides, and it is verified through in vitro and in vivo experiments that all the 5 peptides have antihypertensive effects, and the PFPRP polypeptide has the highest antihypertensive activity, and can be used for the development of antihypertensive drug raw materials.

[0077] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A polypeptide having blood pressure lowering activity, characterized in that: Its N and C termini are aromatic amino acids and proline.

2. The polypeptide having blood pressure lowering activity according to claim 1, characterized in that The polypeptide comprises 3-5 amino acid sequences.

3. The polypeptide having blood pressure lowering activity according to claim 1, characterized in that The polypeptide is any one of the five polypeptides whose amino acids are FGPW, PFPRP, PFA, NPF and FPPP respectively.

4. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of a drug for treating hypotensive disorders.

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