Derivative polypeptide PM6 and application thereof in antithrombosis

Abstract

The invention belongs to the technical field of polypeptide synthesis and biological medicine, and particularly relates to a derived polypeptide PM6 and application thereof in antithrombosis. An original polypeptide SEQ ID NO: 1 is obtained from a transcriptome of salivary glands of poecilobdella manillensis, a truncated polypeptide shown as SEQ ID NO: 3 is obtained from the original polypeptide, and on the basis, {d-Phe}-PRP is added at the N end of the intercepted amino acid sequence through a synthesis strategy of a hirudin analogue Bivalirudin medicine, so that the hirudin analogue is obtained. And connecting {d-Phe}-PRP with the truncated polypeptide through a connecting polypeptide to obtain the derivative polypeptide PM6. The derivative polypeptide PM6 has a good antithrombotic effect, and the bleeding risk of the derivative polypeptide PM6 is equivalent to that of a positive drug, namely, ruvapine.

Description

Technical Field

[0001] The present invention belongs to the field of polypeptide synthesis and biomedicine technology, and specifically relates to a derivative polypeptide PM6 and its application in anti-thrombosis. Background Art

[0002] Thrombosis refers to a small piece of blood formed on the surface of the peeling or repaired inner surface of the blood vessels in the cardiovascular system. It is composed of insoluble fibrin, deposited platelets, and accumulated white blood cells and red blood cells. Under normal circumstances, blood itself has coagulability, which helps the human body to play a role in local coagulation and hemostasis in cases of trauma and bleeding. However, if the balance of the coagulation-anticoagulation mechanism is destroyed during the coagulation process, thrombosis can be formed. Thrombotic diseases such as deep vein thrombosis (DVT), pulmonary embolism (PE) and arterial thrombosis (such as myocardial infarction and stroke) have become one of the types of diseases that the world focuses on.

[0003] Hirudin secreted by leeches is a powerful anticoagulant that can effectively inhibit the activity of thrombin and prevent blood coagulation. It binds to thrombin and prevents it from converting fibrinogen into fibrin, thereby delaying the blood coagulation process. However, some existing studies have found that hirudin has a bleeding risk, which limits its clinical application. Therefore, finding a substance that can replace the function of hirudin and has a low bleeding risk has become an urgent problem to be solved in this field. Summary of the invention

[0004] The purpose of the present invention is to provide a derivative polypeptide PM6 and its application in anti-thrombosis, wherein the derivative polypeptide PM6 has a significant anti-thrombotic effect and the bleeding risk is equivalent to that of the positive drug biluvadin.

[0005] The present invention provides a derivative polypeptide PM6, which is derived from the truncated polypeptide shown in SEQ ID NO: 3 through a synthetic strategy of bivalirudin.

[0006] Preferably, the derived polypeptide PM6 comprises {d-Phe}-PRP, a connecting polypeptide and a truncated polypeptide connected in sequence from the N-terminus to the C-terminus; the amino acid sequence of the truncated polypeptide is shown in SEQ ID NO:3.

[0007] Preferably, the amino acid sequence of the connecting polypeptide is as shown in SEQ ID NO:4.

[0008] The present invention also provides the use of the derivative polypeptide PM6 described in the above technical solution in the preparation of anti-thrombotic products.

[0009] Preferably, the anti-thrombotic product comprises an anti-arterial thrombotic product.

[0010] Preferably, the anti-arterial thrombosis product includes an anti-carotid artery thrombosis product.

[0011] Preferably, the anti-thrombotic product includes a product having the function of inhibiting thrombin activity and / or preventing and treating cerebral hemorrhage.

[0012] Preferably, the product comprises a pharmaceutical product.

[0013] The present invention also provides an anti-thrombotic drug, the active ingredient of which includes the derivative polypeptide PM6 described in the above technical solution.

[0014] Preferably, the only active ingredient in the anti-thrombotic drug is the derivative polypeptide PM6.

[0015] Preferably, the antithrombotic drug comprises a pharmaceutically acceptable excipient.

[0016] Beneficial effects:

[0017] The present invention provides a derivative polypeptide PM6, which is derived from the truncated polypeptide shown in SEQ ID NO:3 through the synthesis strategy of bivalirudin. The present invention obtains the original polypeptide SEQ ID NO:1 from the transcriptome of the salivary gland of the leech, and obtains the truncated polypeptide shown in SEQ ID NO:3 on the original polypeptide. On this basis, {d-Phe}-PRP is added to the N-terminus of the truncated amino acid sequence through the synthesis strategy of the hirudin analog Bivalirudin drug, and the {d-Phe}-PRP is connected to the truncated polypeptide through a connecting polypeptide to obtain the derivative polypeptide PM6. The derivative polypeptide PM6 of the present invention has a good anti-thrombotic effect, and the risk of bleeding is equivalent to that of the positive drug bivalirudin. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0019] Figures 1-2 The results of the determination of the inhibitory activity of the derivative polypeptide PM6 on thrombin in Example 2;

[0020] Figure 3 The results of the determination of the thrombin inhibition constant of the derivative polypeptide PM6 in Example 3;

[0021] Figure 4 The results of the inhibition of the activated partial thromboplastin time (APTT) by the derivative polypeptide PM6 in Example 4 are shown in FIG.

[0022] Figure 5 The results of the determination of the inhibitory effect of the derivative polypeptide PM6 on prothrombin time (PT) in Example 4;

[0023] Figure 6 The results of the determination of the inhibitory effect of the derivative polypeptide PM6 on FeCl3-induced carotid artery thrombosis in Example 5;

[0024] Figure 7 This is a graph showing the bleeding risk detection results of the derived polypeptide PM6 in Example 6. DETAILED DESCRIPTION

[0025] The present invention provides a derivative polypeptide PM6, which is derived from the truncated polypeptide shown in SEQ ID NO: 3 through a synthetic strategy of bivalirudin.

[0026] The present invention obtains the original polypeptide SEQ ID NO:1 from the transcriptome of the salivary gland of the Philippine leech, specifically SVSSENISKMFSLKLFGVFLAVCICVSQAMRYTACTESGQNQCICEGNDVCGQ GRNCQFDSSGKKCVEGEGTHKPQNKGQNDFEPIPEDYFS. On the basis of the original polypeptide, a part of the peptide segment is intercepted to obtain a truncated polypeptide, and the amino acid sequence of the truncated polypeptide is shown in SEQ ID NO:3, specifically: NDFEPIPEDYFS; the truncated polypeptide can bind to the fibrinogen binding site of thrombin and thus interfere with the activity of thrombin. On this basis, the present invention synthesizes the derivative polypeptide PM6 through the synthesis strategy of the hirudin analog Bivalirudin drug (reference [Warkentin, TE, A. Greinacher, and A. Koster, Bivalirudin. Thromb Haemost, 2008.99(5): p.830-9.]), specifically: two fragments {d-Phe}-PRP are added to the N-terminus of the NDFEPIPEDYFS amino acid fragment, and the {d-Phe}-PRP can specifically bind to thrombin and the protease cleavage site between RP, so the polypeptide is easy to clear in the body, the action is reversible, and the risk of bleeding is low. Finally, {d-Phe}-PRP and NDFEPIPEDYFS are connected with a connecting polypeptide to form a derivative polypeptide PM6; as an embodiment, the amino acid sequence of the connecting polypeptide is as shown in SEQ ID NO:4, specifically GGGG; when the amino acid sequence of the connecting polypeptide is GGGG, the amino acid sequence of the derivative polypeptide PM6 is specifically {d-Phe}-PRPGGGGNDFEPIPEDYFS, wherein the amino acid sequence of PRPGGGGNDFEPIPEDYFS is numbered as SEQ ID NO:2. The antithrombotic polypeptide of the present invention not only has the effect of inhibiting thrombin activity, reversible activity, and a relatively short half-life to achieve an antithrombotic effect, but also has a bleeding risk comparable to that of the positive drug bivalirudin; at the same time, the derivative polypeptide PM6 sequence is relatively short and easy to chemically synthesize.

[0027] Based on the above advantages, the present invention also provides the use of the derivative polypeptide PM6 described in the above technical solution in the preparation of anti-thrombotic products. As an embodiment, the anti-thrombotic product can be a product for anti-arterial thrombosis; as another embodiment, the anti-arterial thrombotic product can be a product for anti-carotid artery thrombosis. As an embodiment, the anti-thrombotic product can be a product with the function of inhibiting thrombin activity and / or preventing and treating cerebral hemorrhage. As an embodiment, the product includes a medicine.

[0028] The present invention also provides an antithrombotic drug, the active ingredient of which includes the derivative polypeptide PM6 described in the above technical solution. As an embodiment, the only active ingredient in the antithrombotic drug is the derivative polypeptide PM6. As an embodiment, the antithrombotic drug includes a pharmaceutically acceptable excipient. The present invention does not specifically limit the type of the excipient, and it can be conventionally selected according to the dosage form of the drug.

[0029] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0030] In the following examples, the solvents used to dissolve the derivative polypeptide PM6 and bivalirudin were both physiological saline.

[0031] Example 1

[0032] A derivative peptide PM6, {d-Phe}-PRPGGGGNDFEPIPEDYFS, was obtained by Hangzhou Gutuo Biotechnology Co., Ltd. through solid phase synthesis.

[0033] Example 2

[0034] The inhibitory activity of the derivative polypeptide PM6 on thrombin in Example 1 was determined as follows:

[0035] The inhibitory activity of the derived peptide PM6 on α-thrombin was determined using a luminescent substrate. The derived peptide PM6 (0-500 μg / mL, specifically 0, 4, 20, 100 and 500 μg / mL) was pre-incubated with α-thrombin (50 nM, Enzyme Research Laboratories) at 37°C for 10 min, and then 0.5 mM substrate (S-2238, Chromogenix) was added to a 96-well plate to initiate the reaction. The reaction was continuously monitored at a wavelength of 405 nm for 30 min using an Epoch microplate reader (BioTek) and recorded. The results are shown in Figure 2. Figure 1 and Figure 2 As shown, in Figure 1 A t and A0 represent the absorbance value at a certain time point and the initial absorbance value, respectively. Figure 2 In the vertical axis, A 30 and A0 represent the absorbance value at 30 min and the initial absorbance value, respectively. Figure 2 “*”, “**” and “***” indicate significant differences (P<0.05), P<0.01 and P<0.001, respectively.

[0036] Depend on Figure 1 and Figure 2It can be concluded that the derivative peptide PM6 can inhibit the activity of thrombin in a concentration-dependent manner.

[0037] Example 3

[0038] The determination of the thrombin inhibition constant of the derivative polypeptide PM6 in Example 1 is as follows:

[0039] To calculate the inhibitory constant Ki of the derivative peptide against thrombin, the Dixon plot method was used, with the peptide concentration as the horizontal axis and the inverse of the reaction rate as the vertical axis. Two curves were fitted by regression analysis, and the x value at the intersection of the two curves was calculated as the inhibitory constant of the peptide against thrombin. The reaction rate was obtained by detecting the inhibitory activity curve of the derivative peptide PM6 against thrombin at 405 nm under different concentrations (50 μg / mL and 100 μg / mL) of the luminescent substrate (S-2238). The results are as follows Figure 3 shown.

[0040] Depend on Figure 3 It can be concluded that the derivative peptide PM6 has a strong inhibitory effect on thrombin, and the inhibition constant (Ki) is 1.15 μg / mL (523.2 nM).

[0041] Example 4

[0042] The inhibitory effect of the derivative polypeptide PM6 in Example 1 on activated partial thromboplastin time (APTT) and prothrombin time (PT) was tested in the following steps:

[0043] Coagulation function tests included APTT and PT tests. The absorbance was measured at 650 nm using kits (TC0306, Leagene, for APTT and GMS10176, Genmed, for PT) according to the manufacturer's instructions. The results were as follows: Figure 4 and Figure 5 shown.

[0044] Depend on Figure 4 and Figure 5 It can be concluded that the derived peptide PM6 can inhibit the intrinsic coagulation pathway and the extrinsic coagulation pathway respectively.

[0045] Example 5

[0046] The inhibitory effect of the derivative polypeptide PM6 in Example 1 on FeCl3-induced carotid artery thrombosis is as follows:

[0047] Different dosing groups, including the derivative polypeptide PM6 (1, 0.1 and 0.05 mg / kg), the saline group (negative control) and the bivalirudin group (1 mg / kg, positive control), were injected through the tail vein 10 minutes before surgery. Male C57BL / 6 mice (6-8 weeks) were anesthetized with sodium pentobarbital (80 mg / kg) and the surgical procedure was kept on a heating pad. The neck of the mouse was incised to expose the carotid artery, which was then separated from the vagus nerve and surrounding tissues. After a 2×2mm filter paper filled with 10% ferric chloride solution was attached to the exposed carotid artery, the mouse developed thrombus, and a Doppler microvascular probe (RWD) was placed on the exposed artery to measure vascular blood flow. The results are shown in Figure 2. Figure 6 shown.

[0048] Depend on Figure 6 It can be concluded that the derivative polypeptide PM6 has a significant inhibitory effect on FeCl3-induced carotid artery thrombosis, which further indicates that the derivative polypeptide PM6 has good resistance to carotid artery thrombosis.

[0049] Example 6

[0050] The bleeding risk detection of the derived polypeptide PM6 in Example 1 is as follows:

[0051] Tail bleeding activity assay: Male C57BL / 6J mice (6-8 weeks) were injected with different doses of the derivative peptide PM6 (10, 2 and 0.4 mg / kg) through the tail 10 minutes before surgery, with saline as a negative control and bivalirudin (10, 2 and 0.4 mg / kg) as a positive control. Subsequently, 2 mm was cut off from the tail and carefully immersed in 20 mL of 37°C saline, and the bleeding time was recorded until the bleeding stopped. If bleeding occurred again within 30 seconds, this time would also be counted as the bleeding time in this example. The results are shown in Table 1. Figure 7 As shown in the left picture.

[0052] This example also measured the hemoglobin content of blood collected in 37°C constant temperature physiological saline. The blood collected from each group (200 μL) was incubated with 20 μL 1% (v / v) TritonX-100 (Abcone) at 37°C for 3 min. After incubation, centrifugation was performed at 1000 rpm for 5 min, and the absorbance of the supernatant was measured at 540 nm. The results are shown in Table 1. Figure 7 As shown in the picture on the right.

[0053] Depend on Figure 7 The results in the literature show that the bleeding risk of the derivative peptide PM6 is comparable to that of the positive control group bivalirudin.

[0054] It can be concluded from the above examples that the derivative polypeptide PM6 of the present invention has a significant anti-thrombotic effect, and the bleeding risk is equivalent to that of the positive drug bile duct.

[0055] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A derivative polypeptide PM6, characterized in that: The derived polypeptide PM6 is derived from the truncated polypeptide shown in SEQ ID NO: 3 through a synthetic strategy of bivalirudin.

2. The derivative polypeptide PM6 according to claim 1, characterized in that The derived polypeptide PM6 includes {d-Phe}-PRP, a connecting polypeptide and a truncated polypeptide connected in sequence from the N-terminus to the C-terminus; the amino acid sequence of the truncated polypeptide is shown in SEQ ID NO:

3.

3. The derivative polypeptide PM6 according to claim 2, characterized in that The amino acid sequence of the connecting polypeptide is shown in SEQ ID NO:

4.

4. Use of the derivative polypeptide PM6 according to any one of claims 1 to 3 in the preparation of anti-thrombotic products.

5. The use according to claim 4, characterized in that The anti-thrombotic products include anti-arterial thrombotic products.

6. The use according to claim 4, characterized in that The anti-thrombotic products include products that have the function of inhibiting thrombin activity and / or preventing and treating cerebral hemorrhage.

7. The use according to any one of claims 4 to 6, characterized in that: The products include pharmaceutical products.

8. An antithrombotic drug, characterized in that: The active ingredient comprises the derivative polypeptide PM6 according to any one of claims 1 to 3.

9. The antithrombotic drug according to claim 8, characterized in that: The only active ingredient in the antithrombotic drug is the derivative polypeptide PM6.

10. The antithrombotic drug according to claim 8 or 9, characterized in that: The antithrombotic drug comprises pharmaceutically acceptable excipients.

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