A polypeptide having blood pressure-lowering activity and uses thereof

Abstract

The application discloses a polypeptide with blood pressure lowering activity, which has aromatic amino acid and proline as N and C terminals. The polypeptide comprises 3-5 amino acid sequences. The polypeptide is any one of five polypeptides with amino acids FGPW, PFPRP, PFA, NPF and FPPP. The application also provides application of the polypeptide in a blood pressure lowering drug. The application designs five polypeptides with aromatic amino acid and proline as N and C terminals, simulates the interaction between a peptide segment and ACE, and the polypeptide has good blood pressure lowering effect and is non-toxic, and can be used for development of a blood pressure lowering drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of polypeptides for antihypertensive treatment. More particularly, the present application relates to a polypeptide having antihypertensive activity and use thereof. BACKGROUND

[0002] Hypertension is an important factor leading to diseases such as stroke, coronary heart disease, heart failure, etc. As a chronic disease, most patients need to take antihypertensive drugs for a long time or for life. According to the Clinical Drug Guide of the People's Republic of China, antihypertensive drugs are divided into seven categories, but each category of drugs has different side effects. With the development and maturity of polypeptide synthesis technology, polypeptide drugs have become one of the hotspots of drug research and development. They have been widely used in the prevention, diagnosis and treatment of diseases such as tumors, cardiovascular and cerebrovascular diseases, hepatitis, diabetes, AIDS, etc. due to their wide indications, high safety and significant efficacy, and have broad development prospects. SUMMARY

[0003] In order to achieve these objects and other advantages of the present application, one aspect of the present application provides a polypeptide having antihypertensive activity, wherein the N and C terminal ends are aromatic amino acids and proline.

[0004] According to a preferred embodiment of the present application, the polypeptide comprises 3-5 amino acid sequences.

[0005] According to a preferred embodiment of the present application, the polypeptide is any one of the five polypeptides with amino acids FGPW, PFPRP, PFA, NPF, and FPPP, respectively.

[0006] Another aspect of the present application provides the use of the polypeptide in the preparation of an antihypertensive drug.

[0007] The present application at least has the following advantages: the present application designs five polypeptides with N and C terminal ends being aromatic amino acids and proline, simulates the interaction of the peptide segment with ACE, and proves through in vitro experiments and animal experiments that the five polypeptides all have antihypertensive efficacy, and the polypeptide with the highest antihypertensive activity is PFPRP. The polypeptide of the present application has good antihypertensive effect and is non-toxic, and can be used for the development of antihypertensive drugs.

[0008] Other advantages, objects, and features of the present application will be apparent from the following specification and claims BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 Docking site and details of FGPW and ACE.

[0010] Figure 2The docking site and details of PFPRP and ACE.

[0011] Figure 3 The docking site and details of PFA and ACE.

[0012] Figure 4 The docking site and details of NPF and ACE.

[0013] Figure 5 The docking site and details of FPPP and ACE. DETAILED DESCRIPTION

[0014] The application will be further described below in conjunction with the accompanying drawings, so that those skilled in the art can implement the application according to the description and the drawings.

[0015] The following description is provided to enable any person skilled in the art to make and use the application. The preferred embodiments in the following description are only examples of the application and modifications can be made by those skilled in the art within the spirit and scope of the application. The basic principles defined in the following description can be applied to other embodiments, modifications, improvements, equivalents and other technical solutions without departing from the spirit and scope of the application.

[0016] Test 1: Simulation of the interaction of polypeptides with angiotensin converting enzyme (ACE)

[0017] According to the principle of aromatic amino acids and proline at the N and C termini, five polypeptides were designed, with 3-5 amino acids, and the number of designed polypeptides was 5, namely FGPW, PFPRP, PFA, NPF and FPPP.

[0018] The crystal structure of angiotensin converting enzyme (ACE) (PDB code: 108A) was obtained from the RCSB database (http: / / www.rcsb.org / ), and semi-flexible molecular docking was performed on the five polypeptides using AutoDock 4.2.6 software. Table 1 shows the active peptide sequence and the molecular docking score.

[0019] Table 1: Polypeptide sequence and molecular docking score

[0020] Number Polypeptide Intermolecular force / Kcal 1 FGPW -3.43 2 PFPRP -3.28 3 PFA -2.6 4 NPF -2.56 5 FPPP -2.94

[0021] The five designed polypeptides were subjected to semi-flexible docking with angiotensin converting enzyme (ACE), and all five polypeptides could form stable conformations with ACE, with intermolecular forces including hydrogen bonds, van der Waals forces, etc.

[0022] FGPW polypeptide was subjected to semi-flexible docking with angiotensin converting enzyme (ACE), and the key amino acid residue ARG-501 for interaction with ACE was hydrogen bond.

[0023] PFPRP peptides undergo semi-flexible docking with angiotensin-converting enzyme (ACE). The key amino acid residues that interact with ACE are GLU-176, ALA-261, GLN-262, and HIS-263, and the interaction force is van der Waals force.

[0024] The PFA peptide undergoes a semi-flexible docking with angiotensin-converting enzyme (ACE). The key amino acid residue GLU-176 that interacts with ACE is a hydrogen bond.

[0025] The NPF peptide undergoes a semi-flexible docking with angiotensin-converting enzyme (ACE). The key amino acid residue GLU-176 that interacts with ACE is a hydrogen bond.

[0026] The FPPP polypeptide undergoes a semi-flexible docking with angiotensin-converting enzyme (ACE). The key amino acid residues HIS-263 and ASN-265 that interact with ACE form hydrogen bonds.

[0027] Experiment 2

[0028] The physicochemical properties of five peptides, FGPW, PFPRP, PFA, NPF, and FPPP, were predicted using relevant software. ToxinPred (ToxinPred (iiitd.edu.in)) was used for toxicity and steric hindrance prediction, ExPasy (Expasy - ProtParam) was used for overall average hydrophilicity prediction, admetSAR (admetSAR @ LMMD (ecust.edu.cn)) was used for human intestinal absorption prediction, and pepdraw (PepD http: / / www.pepdraw.com / ) was used for isoelectric point prediction.

[0029] All five antihypertensive peptides are non-toxic and have low steric hindrance, which is conducive to their binding to ACE. Four peptides, FGPW, PFA, NPF, and FPPP, have isoelectric points less than 7, indicating acidity. PFPRP has an isoelectric point of 11.56, greater than 7, indicating alkalinity. PFA has a positive overall average hydrophilicity, indicating strong hydrophobicity and insolubility in water. PGPW, PFPRP, NPF, and FPPP have a negative overall average hydrophilicity, indicating strong hydrophilicity and water solubility. PFA and FPPP show high intestinal absorption, while FGPW, PFPRP, and NPF show poor intestinal absorption. The prediction of these physicochemical properties is of reference value for subsequent peptide modification and drug formulation development.

[0030] Table 2 Predicted Physicochemical Properties

[0031] Peptide sequence Molecular weight Toxicity Isoelectric point Total average hydrophilicity Human intestinal absorption Steric hindrance FGPW 505.62 Non-toxic 5.62 -1.12 - 0.56 PFPRP 612.78 Non-toxic 11.56 -1.30 - 0.49 PFA 333.41 Non-toxic 5.65 1.00 ＋ 0.53 NPF 376.44 Non-toxic 5.27 -0.77 - 0.61 FPPP 456.58 Non-toxic 5.19 -0.50 ＋ 0.44

[0032] Example 3 Hypotensive activity in vitro

[0033] 3.1 The third-party company (Suzhou Modenovo Biotech Co., Ltd.) was entrusted to prepare five screening antihypertensive peptides by Fmoc solid-phase synthesis method, and the purity of the peptides was determined to be greater than 95% by high performance liquid chromatography and mass spectrometry.

[0034] 3.2 Detection of angiotensin converting enzyme inhibitory activity

[0035] The kit was detected by ultraviolet spectrophotometry (Solabao), and the reagents were checked according to the kit instructions, which were reagent one, reagent two, reagent three, and reagent four, of which reagent four was the positive control. According to the instructions, reagent three working solution was prepared, and before use, one was dissolved in 50 uL of distilled water. According to the sample amount, reagent three: reagent one = 9 uL

[0036] : 1791 uL, which was prepared immediately before use, to obtain reagent three working solution.

[0037] FGPW, PFPRP, PFA, NPF, FPPP were respectively configured into concentration gradients of 0.5 mmol / L, 1 mmol / L, 2 mmol / L, 4 mmol / L, 8 mmol / L, 12 mmol / L, 16 mmol / L, and 20 mmol / L. Among them, FGPW, PFPRP, NPF, FPPP were dissolved and diluted to the corresponding concentration with reagent one provided in the kit, and PFA was dissolved in 1% ethanol and then diluted to the corresponding concentration with reagent one. The obtained samples of different concentrations were respectively placed in the determination tube, 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).

[0038] Table 3

[0039]

[0040] ACE inhibitor inhibition rate (%) = (ΔA 空白 -ΔA 测定 ) ÷ ΔA 空白 × 100% (1).

[0041] IC 50 value calculation: with sample concentration as abscissa and inhibition rate as ordinate, an inhibition curve was drawn, and the sample concentration when the inhibition rate was 50% was obtained.

[0042] The IC 50 values of the five antihypertensive peptides are shown in Table 5.

[0043] Table 4 IC 50 values of the five antihypertensive peptides

[0044] Peptide sequence IC 50 (μmol / L) FGPW 5.46 PFPRP 2.51 PFA 7.28 NPF 12.37 FPPP 8.36

[0045] From the data of Table 4, it can be seen that the five polypeptides all have in vitro antihypertensive activity, among which the IC 50 of the PFPRP polypeptide is the lowest, 2.51 mmol / L, and the in vitro antihypertensive activity is the highest. The IC 50 value refers to the inhibitor concentration when the ACE inhibition rate reaches 50%, and it is a very accurate index for evaluating the in vitro inhibitory activity of ACE inhibitory peptides. The smaller the IC 50 value, the stronger the activity of the ACE inhibitory peptide. According to the ACE inhibitory activity peptides reported in the literature, the IC 50 values of the five polypeptides provided in the present application are all below 20 µmol / L, which can exhibit significant antihypertensive effect. Thus, it can be seen that the five short peptides provided in the present application all exhibit very strong in vitro antihypertensive activity, and the in vitro antihypertensive activity of the PFPRP polypeptide is the highest.

[0046] 3.3 In vivo antihypertensive effect experiment of the antihypertensive peptides

[0047] A third-party company (Suzhou Modif Biological Technology Co., Ltd.) was commissioned to prepare the five screened antihypertensive peptides by Fmoc solid-phase synthesis method, and the peptide purity was determined to be greater than 95% by high performance liquid chromatography and mass spectrometry analysis. In the animal experiment, the polypeptide group drugs were all prepared from the synthesized polypeptide solid powder.

[0048] Seventy spontaneously hypertensive rats (SHR), male, 10~12 weeks old, weighing 210~230g; ten normal blood pressure rats (WKY), male, 10~12 weeks old, weighing 210~230g. The feeding environment temperature was maintained at (25±1)℃, the humidity was maintained at 55%±5%, the light / dark cycle was 12 hours per day, the lighting time was from 8:00 am to 20:00 pm, the rats could freely eat standard feed and tap water, and the feeding environment was disinfected regularly. Before the experiment, the SHR and WKY had a one-week environmental adaptation period. After the adaptation period, the body weight and blood pressure of the rats were measured, and according to the measurement results, they were divided into different groups to ensure that the baseline data of the rats in each group were uniform.

[0049] The SHR rats were divided into 7 groups, 10 rats in each group, which were respectively a captopril intervention positive control group, an FGPW polypeptide intervention group, a PFPRP polypeptide intervention group, a PFA polypeptide intervention group, an NPF polypeptide intervention group, an FPPP polypeptide intervention group, a hypertensive model control group, and 10 WKY rats as a blank control group.

[0050] Table 5 Dose table for each group

[0051] Group intraperitoneal injection / mg.kg -1 ]] Blank control group Equal volume of normal saline Hypertension model control group Equal volume of normal saline FGPW polypeptide intervention group FGPW normal saline dissolution preparation 20 mg / kg PFPRP polypeptide intervention group PFPRP normal saline dissolution preparation 20 mg / kg PFA polypeptide intervention group PFA normal saline dilution preparation 20 mg / kg NPF polypeptide intervention group NPF normal saline dissolution preparation 20 mg / kg FPPP polypeptide intervention group FPPP normal saline dissolution preparation 20 mg / kg Positive control group 10 mg / kg Captopril

[0052] Common feed, according to the dose of gavage table daily gavage administration 1 times, continuous gavage 8 weeks. During the experiment, the body weight, food intake and blood pressure of rats were collected every week.

[0053] When measuring the blood pressure and heart rate of rats, in order to ensure the accuracy of the measured data, the rats to be measured need to be placed in a warm box (37℃) in advance and left for about 15 min. After the rats are calm and the blood pressure is stable, the systolic pressure, diastolic pressure and heart rate of SHR and WKY rats are monitored using BP-2006A small animal noninvasive blood pressure instrument. The rats are fixed in the heating cover, and at the same time, in order to avoid the stress reaction of the rats affecting the accuracy of the test data, a light shield is wrapped outside the heating cover. The rats are placed in the heating cover and covered with a light shield, and the temperature of the heating cover is set to 37~39℃ to ensure that the tail artery of each rat is fully dilated during measurement. The blood pressure cuff is worn on the tail root of the rat 3 cm away from the fixed position, and at the same time, the "pulse signal sensor" in the cuff is kept above the tail artery. When the sensor shows a stable wavy pulse wave, blood pressure measurement begins. The system is connected to a computer, and the test results are recorded by BP98AWU software. The heart rate changes less than 10 times / min, and the blood pressure changes less than 20 mmHg. Three to five readings are recorded, and each rat is measured three times to get the average value.

[0054] Each group was gavaged for 8 weeks, and the growth and development, activity of animals in each group were normal during the experiment, and the mental state was good, without toxic manifestations and death.

[0055] Table 6 Change of body weight of rats in each group

[0056]

[0057] Table 6 shows the body weight changes of rats in each group after 1 week, 4 weeks and 8 weeks of administration. There is no significant change in body weight among groups, and the growth and development, activity of animals in each group are normal during the experiment, and the mental state is good, without toxic manifestations and death. It shows that the 5 kinds of antihypertensive peptides screened have no toxic side effects.

[0058] Table 7 Systolic pressure (SBP) of rats in each group after 8 weeks of intervention

[0059]

[0060] Table 7 shows the detection values of systolic pressure of rats in each group every week. In the 0th week of intervention, the diastolic pressure of the hypertensive model group, each polypeptide group and the positive control group is significantly higher than that of the blank control group (P<0.05), and the hypertensive model is established.

[0061] After 8 weeks of intervention, the blood pressure of the polypeptide group and the positive control group compared with the hypertension model group decreased to a certain extent. The FGPW polypeptide group, the PFPRP polypeptide group and the positive control group significantly decreased compared with the hypertension model group (P<0.05), and the PFPRP polypeptide group had the largest decrease, which was equivalent to the positive control group.

[0062] Table 8 Diastolic blood pressure (DBP) of rats in each group after 8 weeks of intervention

[0063]

[0064] Table 8 is the detection value of the diastolic blood pressure of rats in each group every week. In the 0th week of intervention, the diastolic blood pressure of the hypertension model group and 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.

[0065] In the 8th week of intervention, the diastolic blood pressure of the polypeptide groups and the positive drug group compared with the hypertension model control group decreased. The diastolic blood pressure of the FGPW polypeptide group and the PFPRP polypeptide group was significantly lower than that of the hypertension model group (P<0.05), and the PFPRP polypeptide group had the largest decrease, and the decrease rate was equivalent to that of the positive control group.

[0066] Table 9 Heart rate (HR) of rats in each group after 8 weeks of intervention

[0067]

[0068] Table 9 is the detection value of the heart rate of rats in each group after 8 weeks of intervention. The heart rate of the hypertension model control group compared with the blank control group significantly increased (P<0.05), and the heart rate of the polypeptide groups and the positive control group compared with the hypertension model control group significantly decreased (P<0.05), and the heart rate decreased obviously.

[0069] Combined with the half-inhibition rate detection result of the ACE inhibitor and the antihypertensive peptide in vivo, the five screened antihypertensive peptides all have antihypertensive effect, and the antihypertensive effect of the FGPW and PFPRP polypeptides is obviously better than that of the other three polypeptides, and the dose efficacy result of the PFPRP polypeptide 20mg / kg is equivalent to that of the positive drug (captopril) 10mg / kg.

[0070] The application designs five antihypertensive peptides, and the five peptides are verified to have antihypertensive effect through in vitro and in vivo experiments, and the PFPRP polypeptide has the highest antihypertensive activity, and can be used for the development of antihypertensive drug raw materials.

[0071] While embodiments of the application have been disclosed in connection with the above specification and drawings this description is not intended to limit the scope of the application and many modifications, enhancements, alternatives, and variations will become apparent to those skilled in the art from this disclosure. Accordingly, it is intended that the application not be limited to the described embodiments, but that it include all variations falling within the scope of the claims, and their equivalents.

Claims

1. The application of a polypeptide in the preparation of antihypertensive drugs, characterized in that, The amino acid sequence of the polypeptide is any one of FGPW, PFPRP, PFA, NPF, and FPPP.

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