CN117143194B - Anti-platelet aggregation polypeptide, preparation method and application thereof - Google Patents

Abstract

The invention discloses an anti-platelet aggregation polypeptide, a preparation method and application thereof. Based on a large number of screening experiments, the invention discovers that the specific polypeptide can be combined with the PEAR1 receptor on the surface of the blood platelet, thereby inhibiting or antagonizing the aggregation function of the blood platelet, and being used as an anti-blood platelet medicament for preventing and treating thrombus and embolism diseases.

Description

Anti-platelet aggregation polypeptide, preparation method and application thereof

Technical field

The present invention relates to the field of anti-platelet aggregation, specifically to anti-platelet aggregation polypeptides targeting PEAR1 receptors, their preparation methods and applications.

Background technique

Antiplatelet drugs can inhibit platelet aggregation and are one of the important drugs for the prevention and treatment of thromboembolic diseases. In patients with transient ischemic attack or ischemic stroke, antiplatelet drug therapy during the acute and recovery phases can significantly improve patient prognosis. In patients with acute coronary syndrome after percutaneous coronary intervention, antiplatelet drug therapy can reduce the risk of adverse cardiovascular events. Therefore, antiplatelet drugs are not only important therapeutic drugs for thromboembolic diseases, but also secondary preventive drugs for many cardiovascular diseases, playing an important clinical role.

Currently, the antiplatelet drugs used clinically in China mainly target three receptors: cyclooxygenase, P2Y ₁₂ receptor, and glycoprotein IIb/IIIa (GPIIb/IIIa) receptor. Aspirin is a cyclooxygenase antagonist that blocks the synthesis of thromboxane A2 by antagonizing cyclooxygenase, thereby inhibiting platelet aggregation. However, because cyclooxygenase is widely distributed in blood vessels, stomach and kidney tissues, aspirin use can cause adverse reactions such as gastrointestinal tract and asthma. Clopidogrel, prasugrel, and ticagrelor are P2Y ₁₂ receptor antagonists. P2Y ₁₂ receptor antagonists inhibit platelet aggregation by inhibiting the binding of adenosine diphosphate to P2Y ₁₂ receptors. However, individual differences in its clinical application are large, and bleeding or ischemic adverse events may occur during treatment, affecting the clinical prognosis of patients. GPIIb/IIIa receptor antagonists include abciximab, tirofiban, and eptifibatide. Inhibiting GPIIb/IIIa blocks its binding to ligands such as fibrinogen and inhibits platelet aggregation. However, the antiplatelet effect of GPIIb/IIIa receptor is too strong, so it can only be used intravenously for a short period of time.

Therefore, existing drugs have shortcomings such as few available types, large individual differences, adverse reactions, and intolerance. There is an urgent need to discover new targets related to platelet function and develop drugs targeting new targets.

The information in the Background is merely illustrative of the general background of the invention and should not be construed as an admission or in any way implying that the information constitutes the prior art that is already known to those of ordinary skill in the art.

Contents of the invention

In order to solve at least part of the technical problems in the prior art, the present invention provides polypeptides with platelet aggregation-inhibiting activity. Specifically, the present invention includes the following contents.

One aspect of the present invention provides a polypeptide, which has an activity of inhibiting platelet aggregation and has a structure described by the following formula:

V1-LKYX1YENHX2-V2 formula (I);

Among them, X1 represents W or H, X2 represents A or N; V1 does not exist or represents the first variable region, and V2 does not exist or represents the second variable region.

In certain embodiments, the polypeptide according to the invention, wherein the first variable region is selected from A, EA or GEA, and the second variable region is selected from I, IS, ISI.

A second aspect of the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the polypeptide described in the first aspect.

A third aspect of the invention provides a carrier molecule comprising the nucleic acid molecule of the invention.

A fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule described in the second aspect of the present invention or the vector molecule described in the third aspect.

A fifth aspect of the present invention provides a method for preparing the polypeptide according to the first aspect, which includes preparing it through artificial synthesis or through genetic engineering.

A sixth aspect of the present invention provides a pharmaceutical composition comprising the polypeptide described in the first aspect and/or the nucleic acid molecule described in the second aspect, and a pharmaceutically acceptable carrier.

A seventh aspect of the present invention provides the use of polypeptides or nucleic acid molecules thereof in the preparation of anti-platelet aggregation drugs.

An eighth aspect of the present invention provides a method for inhibiting platelet aggregation in vitro, which includes the step of contacting the polypeptide described in the first aspect of the present invention with platelets in vitro.

In view of the current situation that there are no drug molecules targeting the PEAR1 receptor inhibitory drug molecules, the present invention, based on a large number of screening experiments, found that specific polypeptides can bind to the PEAR1 receptor on the surface of platelets, thereby inhibiting or antagonizing the platelet aggregation function. This can As an antiplatelet drug, it is used to prevent and treat thrombosis and embolism diseases.

Description of drawings

Figure 1 shows the inhibition of platelet function corresponding to different polypeptide sequences with the structure shown in formula (I).

Figure 2 is an exemplary polypeptide binding curve to PEAR1.

Detailed ways

Various exemplary embodiments of the invention will now be described in detail. This detailed description should not be construed as limitations of the invention, but rather as a more detailed description of certain aspects, features and embodiments of the invention.

It should be understood that the terms used in the present invention are only used to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges in the present invention, it should be understood that the upper and lower limits of the range and every intermediate value therebetween are specifically disclosed. Every smaller range between any stated value or value intermediate within a stated range and any other stated value or value intermediate within a stated range is also included within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded from the range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and/or materials in connection with which the documents relate. In the event of conflict with any incorporated document, the contents of this specification shall prevail.

As used herein, the term "polypeptide" refers to a polymer composed of multiple amino acid residues, or variants, synthetic or naturally occurring analogs thereof. Thus, polypeptides are suitable for use in amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids (eg, chemical analogs of the corresponding naturally occurring amino acids), as well as to naturally occurring amino acid polymers and their Naturally occurring chemical derivatives. Examples of such derivatives include, but are not limited to, post-translational modifications and degradation products including pyroglutamyl, isoaspartyl, proteolytic, phosphorylated, glycosylated, oxidized, isomerized and deamination variants.

As used herein, the term "platelet aggregation-inhibiting activity", sometimes referred to as "anti-platelet aggregation activity" or "anti-platelet activity", refers to the activity of reducing, reducing, inhibiting or blocking platelet aggregation, preferably with an inhibition rate of more than 5%. , 10% or more, preferably 12% or more, such as 14% or more, 15% or more. The inhibition rate refers to the aggregation amount of platelets in the control group and the aggregation amount of platelets to be tested/the aggregation amount of platelets in the control group when measured under the same conditions. The measurement of the inhibition rate is not particularly limited, and includes using a platelet aggregator (such as Helena AggRAM) to record the platelet aggregation inhibition measurement within 5 minutes.

polypeptide

In one aspect, the present invention provides a polypeptide, which is a short peptide with an activity of inhibiting platelet aggregation. Because of its ability to target the PEAR1 receptor, it is sometimes called a "PEAR1 receptor inhibitor." Generally speaking, the length of the polypeptide of the present invention is less than 20 amino acids, such as less than 18 amino acids, less than 15 amino acids, less than 12 amino acids, etc.

In certain embodiments, a polypeptide of the invention has a structure represented by Formula (I):

V1-LKYX1YENHX2-V2 Formula (I).

In formula (I), X1 represents W or H, X2 represents A or N, V1 does not exist or represents the first variable region, and V2 does not exist or represents the second variable region.

Wherein, the first variable region is selected from A, EA or GEA. The second variable region is selected from I, IS, ISI.

In formula (I), when V1, V2, and V3 do not exist, "-" does not exist. When V1 and V2 represent different amino acid residues, "-" represents a covalent bond, such as a peptide bond.

Exemplarily, the polypeptide of the invention is selected from one of the following sequences:

LKYWYENHA(SEQ ID No.1),

EALKYWYENHA(SEQ ID No.2),

GEALKYWYENHA(SEQ ID No.3),

EALKYHYENHA(SEQ ID No.4),

GEALKYWYENHNI(SEQ ID No.5),

GEALKYWYENHNISI (SEQ ID No. 6).

nucleic acid molecules

One aspect of the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide of the invention.

The present invention is not particularly limited to nucleic acid molecules as long as they can produce the polypeptides of the present invention, which include nucleotide sequences encoding polypeptides and optional other sequences. Exemplary other sequences are operably linked to the nucleotide sequence encoding the polypeptide. The nucleotide sequence encoding a polypeptide may be a nucleotide sequence containing one polypeptide gene or a nucleotide sequence containing multiple polypeptide genes connected in series. Examples of other sequences include, but are not limited to, control sequences for gene expression, such as promoters, any leader functional in the host cell, terminators, and the like. Nucleotide sequences encoding polypeptides include native coding sequences (e.g., sequences readily determined by the skilled artisan using available databases) or degenerate sequences, such as codon-optimized coding sequences designed for a particular host cell, in particular the host A codon-optimized version was performed.

carrier molecule

One aspect of the invention provides a carrier molecule comprising the nucleic acid molecule of the invention.

A vector of the present invention refers to an artificial construct capable of delivering and preferably expressing one or more genes or sequences of interest in a host cell. The vector of the present invention is not limited and may be an expression vector, a viral vector, etc. In certain embodiments, the vector contains a target gene encoding a polypeptide gene of the invention or a precursor thereof, a promoter, a terminator, or optionally further contains a marker gene. As the vector, a known vector or a self-constructed vector can be used. Known vectors include plasmid vectors, lentiviral vectors, adenoviral vectors, AAV viral vectors, etc.

host cell

One aspect of the invention provides a host cell comprising the nucleic acid molecule of the invention or the vector molecule of the invention.

The host cell of the present invention refers to any cell type suitable for transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. A host cell includes any descendant of a parent cell that differs from the parent cell due to mutations that occur during replication.

Preparation

One aspect of the invention provides a method for preparing the polypeptide of the invention. The preparation method is not particularly limited, including preparation by artificial synthesis or genetic engineering.

In certain embodiments, the polypeptides of the invention are obtained synthetically. Methods of artificially synthesizing polypeptides are known in the art. Specifically, it includes two methods: liquid phase and solid phase. Liquid-phase peptide synthesis methods include BOC protection and Z protection. Such methods preferably apply short peptide synthesis, especially the polypeptide synthesis of the present invention. Compared with solid-phase peptide synthesis, it has many advantages such as a wide selection of protecting groups, low cost, and easy scale-up of synthesis. For the polypeptide of the present invention, liquid phase polypeptide synthesis is generally adopted.

In certain embodiments, the polypeptide of the present invention is synthesized by a solid-phase polypeptide synthesis method, which includes the use of FMOC protection and the use of BOC protection. It has the advantages of convenient and rapid synthesis and easy automation.

In certain embodiments, the polypeptides of the invention are obtained by expression through genetic engineering. Genetically engineered expression systems include prokaryotic cell expression systems, eukaryotic cell expression systems and cell line-free expression systems. Examples of prokaryotic cell expression systems include E. coli expression systems. Eukaryotic cell expression systems include enzyme mother expression systems, insect cell expression systems and mammalian cell expression systems.

pharmaceutical composition

One aspect of the present invention provides a pharmaceutical composition comprising the polypeptide and/or nucleic acid molecule of the present invention and a pharmaceutically acceptable carrier. Polypeptides and nucleic acid molecules have been described in detail above and will not be described again here. Pharmaceutically acceptable carriers are described below.

In the present invention, pharmaceutically acceptable carriers are well known in the art, and those of ordinary skill in the art can determine that they meet clinical standards. Pharmaceutically acceptable carriers include diluents and excipients.

Examples of suitable pharmaceutically acceptable carriers include, but are not limited to: (1) Dulbecco's phosphate buffered saline, pH about 7.4, containing or not containing about 1 mg/ml to 25 mg/ml human serum albumin; (2) 0.9% saline (0.9% w/v sodium chloride), and (3) 5% (w/v) glucose; antioxidants such as tryptamines and stabilizers such as Tween20 may also be included.

The pharmaceutical composition of the present invention may be in any suitable dosage form. For example, injections, suspensions, emulsifiers, etc. The pharmaceutical composition of the present invention can be administered into the body by known means. For example, delivery is by intramuscular injection into the tissue of interest, optionally administered via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such administration may be via a single dose or multiple doses. It will be understood by those skilled in the art that the actual dosage to be administered herein may vary depending largely on a variety of factors, such as the target cell, the type of organism or tissue thereof, the general condition of the subject to be treated, the route of administration , administration methods, etc.

use

One aspect of the present invention provides the use of the polypeptide of the present invention or its nucleic acid molecule in the preparation of anti-platelet aggregation drugs.

Currently, existing antiplatelet drugs generally target cyclooxygenase, P2Y ₁₂ receptors, and glycoprotein IIb/IIIa (GPIIb/IIIa) receptors. Different from existing drugs, the polypeptide of the present invention can bind to PEAR1, thereby inhibiting or blocking the activation of the PEAR1 receptor on the platelet surface, and this activation can cause tyrosine phosphorylation of the intracellular segment of PEAR1, resulting in phosphatidylinositol Phosphorylation of -3-kinase causes activation of GPIIb/IIIa and platelet aggregation.

Methods to inhibit platelet aggregation

In one aspect, the present invention provides a method for inhibiting platelet aggregation, which includes the step of contacting the polypeptide of the present invention with platelets. The method of the present invention may be an in vitro method or an in vivo method. Inhibiting platelet aggregation in vitro can be used to analyze or study platelet function, and can then be used, for example, for screening of useful compounds, especially screening of high-energy compounds.

Example 1

1. Experimental instruments and reagents

Experimental instruments: 1/10000 electronic balance; vertical reactor (20*25mm, No. 1 sand core); centrifuge tube; 100-1000µl pipette; 1ml pipette; nitrogen; circulating water vacuum pump; bottle washing ; test tube; long-neck straw;

Experimental reagents: 2-cl resin; Fmoc-AA-OH; anhydrous DCM; DIEA; methanol; DCM; industrial grade DMF; analytical grade DMF; 20% piperidine/DMF; detection reagent A (5g ninhydrin-100ml without Water ethanol), B (analytical grade pyridine); HOBT; DIC;

2. Experimental steps

(1) Weigh 500mg of 2-Cl-Trt resin on an electronic balance, put it into a 25ml reactor, add DCM and soak for 30 minutes. Then wash 3 times with DMF and 1 time with DCM.

(2) Weigh 0.15 mmol of Fmoc-AA-OH (C end) into a centrifuge tube, add 8 ml of anhydrous DMF and 800 µl of DIEA, and shake well. Use a pipette to add the solution to the reactor in the previous step, and react with nitrogen bubbles for 1.5 hours. At the end of the reaction, wash the resin with DMF three times, 30 seconds each time, and add a mixture of 0.5ml DIEA, 0.5ml methanol, and 2ml DCM. , react for 20 minutes.

(3) Washing: After draining the liquid in the reactor with a circulating water vacuum pump, use a washing bottle to add industrial grade DMF to the reactor so that the resin is completely soaked in the solution. Wash for 30 seconds, and then use a circulating water vacuum pump to drain the reactor. Drain the liquid in the container and repeat this operation 4 times.

(4) Remove Fmoc: Use a washing bottle to add 20% piperidine/DMF solution into the reactor. The reagent volume accounts for about 3 times the volume of the resin, so that the resin is completely soaked in the solution, and nitrogen bubbles for 20 minutes.

(5) Washing: Follow step (3), and replace the industrial grade DMF with analytical grade DMF during the fifth washing process;

(6) Resin detection: Use a long-neck straw to take 10-20 resins in the reactor and place them at the bottom of the detection tube. Then use a rubber dropper to take two drops of detection reagents A and B and drop them into the test tube to mix the resin and detection reagents. can fully contact, and then put the test tube into a constant temperature heating of 100°C for 2 minutes.

(7) Condensation: Weigh 0.5mmol Fmoc- AA-OH (the second position of the C-terminal) and 0.5mmol HOBT (0.075g) in a centrifuge tube, fully dissolve it with 1ml DMF; then add 100µl DIC, mix for 1 minute, and add Into the dried resin, nitrogen was bubbled for 1 hour.

(8) Repeat operations (5)-(7) until the peptide connection is completed.

(9) Finally, remove Fmoc, then wash, and then wash the resin three times with methanol according to the washing method. Finally, use a circulating water vacuum pump to drain the liquid in the reactor, and vacuum dry until the resin becomes granular.

Example 2

1. Experimental method:

16-week-old C57BL/6J mice were prepared. After appropriate anesthesia with 20% urethane, the abdominal aorta was extracted using 3.8% sodium citrate anticoagulation (anticoagulation: blood = 1:9). Centrifuge at 90 g for 9 min to collect the upper plasma, centrifuge at 150 g for 15 min, discard the supernatant and add benchtop solution to obtain washed platelets, and adjust the number of platelets in the washed platelets to 3-4×10 ⁸ /mL. Pipette 240 μL of washed platelets, add 5 μL of normal saline to the control group, and add 5 μL of polypeptide (final concentration 1 mg/mL) to the experimental group, and incubate at 37°C for 5 minutes. After adding the magnetic rotors respectively, add 5 μL ADP solution (01905, Sigma: Darmstadt, Germany; final concentration 50 μM), record the platelet aggregation within 5 minutes on a platelet aggregator (Helena AggRAM), and compare the maximum platelets of the experimental group and the control group. aggregation rate.

2. Experimental results:

The results are shown in Figure 1. Figure 1 shows the inhibition of platelet function corresponding to different polypeptide sequences having the structure shown in formula (I). If the platelet inhibition rate is higher than 15%, the polypeptide is considered to have an inhibitory effect on platelet aggregation and has antiplatelet activity. As shown in Figure 1, based on the results of peptides 1, 2, 3, 4, and 5, it was found that LKYWYENHA is a sequence that maintains antiplatelet activity. In order to further determine the lengthening of the front-end and back-end amino acids, polypeptides 6, 7, and 8 were constructed. It was found that the antiplatelet activity of the peptides gradually decreased with the lengthening of the amino acids, and the front-end could be extended by up to three amino acids GEA. Constructs 13, 14, and 15, it was found that the antiplatelet activity of the peptide gradually decreased with the extension of amino acids, and its back end could be extended by up to three amino acids ISI. In order to confirm the variability of amino acids, amino acids at different positions were replaced to form polypeptides 9, 10, 11, and 12. It was found that only the W amino acid could be replaced by the H amino acid and still had antiplatelet activity. Therefore, based on the above experimental results, it is concluded that the polypeptide with the following formula is an active polypeptide:

(G)+(E)+(A)+L+K+Y+W(H)+Y+E+N+H+A(N)+(I)+(S)+(I) Formula (III) ).

Among them, "+" indicates a covalent bond, and "()" indicates that the corresponding amino acid residues in brackets are optional amino acid residues, which may or may not exist.

Example 3

This example is used to test that the polypeptide can bind to the PEAR1 protein. The following takes polypeptide 1 as an example for verification.

1. Experimental method:

The human PEAR1 recombinant protein was immobilized on the Cytiva CM5 chip by surface plasmon resonance using an amino coupling reagent. Peptide 1 was serially diluted using PBS-P, and then the protein solution was flowed through the chip at a flow rate of 30 μL/min for 120 s, followed by dissociation for 480 s. Use glycine hydrochloride solution (pH 2.0) to regenerate the chip surface, and use the 1:1 binding model in the Biaevaluation program to obtain the kinetic data of the compound.

2. Experimental results:

As shown in Figure 2, polypeptide 1 can bind to the PEAR1 protein with a binding constant Kd of 0.17 μM.

Example 4

This example is used to verify that the polypeptide can inhibit platelet function in mice.

1. Experimental method:

Prepare 16-week-old C57BL/6J mice and Pear1 gene knockout mice. The negative control group uses C57BL/6J mice for intravenous injection of normal saline at a rate of 0.05ml/10g, and the experimental group uses C57BL/6J mice at a rate of 1 mg/kg polypeptide 1. 0.05ml/10g was injected intravenously into the orbit. In the positive control group, Pear1 knockout mice were injected intravenously with normal saline at the rate of 0.05ml/10g and waited for 30 minutes. After 30 minutes, ADP 0.05ml/10g was injected into the orbital vein at a dose of 250mg/kg. Observe the survival of the mice within 10 minutes.

2. Experimental results:

The mice in the negative control group quickly showed shortness of breath and difficulty breathing, and 90% (9/10) of the mice died within 10 minutes, with a survival rate of 10%. Polypeptide 1 can increase the survival rate of ADP-induced pulmonary embolism mice to 75% (3/4) after intravenous administration for 30 minutes, and the survival rate of Pear1 gene knockout mice in the positive control group is 75% (9/12).

While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Various modifications or changes may be made to the exemplary embodiments described herein without departing from the scope or spirit of the invention. The scope of the claims should be given the broadest interpretation to cover all modifications and equivalent structures and functions.

Claims (8)

1. A polypeptide characterized in that it has an activity of inhibiting platelet aggregation and is at least one of the amino acid sequences shown in SEQ ID No. 1-6. 2. Nucleic acid molecule, characterized in that it contains a nucleotide sequence encoding the polypeptide according to claim 1. 3. Nucleic acid vector, characterized in that it contains the nucleic acid molecule of claim 2. 4. Host cell, characterized in that it contains the nucleic acid molecule of claim 2 or the nucleic acid vector of claim 3. 5. The method for preparing the polypeptide according to claim 1, characterized in that it is prepared by artificial synthesis or genetic engineering. 6. Pharmaceutical composition, characterized in that it contains the polypeptide according to claim 1 and a pharmaceutically acceptable carrier. 7. Use of the polypeptide according to claim 1 or the nucleic acid molecule according to claim 2 in the preparation of drugs for treating thrombosis. 8. A method for inhibiting platelet aggregation in vitro, comprising the step of contacting the polypeptide according to claim 1 with platelets.