WO2023072311A1 - Whitmania pigra polypeptide against coagulation factor xia and use thereof - Google Patents

Abstract

Provided are a Whitmania pigra polypeptide against coagulation factor XIa and a mutant thereof. A Kunitz-type polypeptide sequence is screened from a salivary gland transcriptome database of Whitmania pigra and subjected to expression construction, screening and amino acid replacement to obtain a series of polypeptides. The polypeptides are used as a prodrug for preventing or treating diseases such as brain injuries (such as strokes and cerebral apoplexy), has the characteristics of safety, effectiveness, etc., and is suitable for the research and development of drugs having an anti-thrombus or anticoagulant activity.

Description

Anticoagulant Factor XIa Polypeptide and Its Application

Cross References to Related Applications

This application claims the benefit of Chinese Patent Application No. 202111261322.8 filed on October 28, 2021, which is hereby incorporated by reference in its entirety.

sequence listing

This application contains a Sequence Listing and is hereby incorporated by reference in its entirety.

technical field

The invention belongs to the field of polypeptides, and relates to the application of the broad-bodied leech anticoagulant factor XIa polypeptide and its mutants. Specifically, the present invention relates to anticoagulant factor XIa polypeptides and mutants thereof that inhibit the activity of coagulation factor XIa, nucleic acids encoding anticoagulant factor XIa polypeptides, expression cassettes/vectors comprising the nucleic acids, and any of the above-mentioned item's cell. The present invention also relates to a pharmaceutical composition comprising the anticoagulant factor XIa polypeptide and its mutants. The present invention further relates to methods of using said peptides or pharmaceutical compositions for the treatment or prevention of diseases associated with thrombosis.

Background technique

With the aging of society, the incidence of cardiovascular and cerebrovascular diseases is increasing year by year. The 2021 "China Cardiovascular Disease Report" shows that in my country, cardiovascular disease accounts for 46.7% of disease deaths, ranking first among various diseases. Cardiovascular diseases mainly include acute coronary syndrome, myocardial infarction, stroke, unstable angina, transient cerebral ischemia and atherosclerosis. Thrombosis is the main cause of cardiovascular disease. Common clinical drugs used to prevent and treat such diseases are rivaroxaban, apixaban, aspirin, clopidogrel, heparin, eptifibatide, etc. However, these drugs all have different degrees of bleeding side effects, which bring risks to clinical treatment. Therefore, it is imperative to develop anticoagulant drugs without bleeding risk.

Coagulation in mammalian plasma involves a large number of proteins, creating a cascade of reactions leading to thrombin generation. Thrombin then converts fibrinogen to fibrin, causing blood to coagulate. The most commonly used clinical index to reflect the coagulation activity of the intrinsic coagulation system is activated partial thromboplastin time (APTT), and the most commonly used index to reflect the coagulation activity of the exogenous coagulation system is prothrombin time (PT).

Clinical research data show that the rate of ischemic stroke and deep vein thrombosis is significantly reduced in patients who are congenitally deficient in coagulation factor XI (factor XI, FXI), and generally have no spontaneous bleeding. Studies have found that FXI can enhance the generation of thrombin in thrombus formation, but has limited effect on the generation of thrombin in the process of hemostasis. Animal experiments have shown that FXI knockout mice have the ability to resist artificially induced arterial and venous thrombosis, and their phenotype and reproductive ability are normal. In the baboon arteriovenous thrombosis model, heparin significantly prolongs APTT, PT time, and bleeding time. The FXI antibody only caused the prolongation of the APTT time, but did not affect the PT time and bleeding time. Based on the above characteristics of FXI/FXIa, FXI/FXIa has become an ideal target for the development of new antithrombotic drugs.

The broad-bodied leech is widely distributed, and its salivary glands contain a variety of antithrombotic active components such as thrombin inhibitor hirudin, FXa inhibitor antistasin and so on. But there is no report about FXI/FXIa inhibitors.

Contents of the invention

The purpose of the present invention is to provide a novel polypeptide for inhibiting FXIa.

Technical solutions

The research team determined the transcriptome of the salivary gland of the wide-bodied golden leech (Whitmania Pigra), established a transcriptome database, screened out multiple Kunitz-type polypeptide sequences, and constructed and expressed them, amino acid substitutions, and obtained a series of polypeptides, named For WPK1-5.

The peptide WPK5 with strong antithrombotic activity was obtained through preliminary screening. In order to improve the antithrombotic effect of WPK5, based on WPK5, the Loop replacement strategy was adopted to improve its activity. Based on the new Kunitz skeleton of WPK5, Loop1 ( ¹¹ TGPCRAMISR ²⁰ ) and Loop2 ( ³⁴ FYGGC ³⁸ ) in the coagulation factor XIa inhibitor PN2KPI were used to replace Loop1 ( ¹¹ TGPRSNLER ²⁰ ) and Loop2 ( ³⁴ QYGGC ³⁸ ) in WPK5, A new polypeptide was obtained and named as WPK5-mut.

The amino acid sequences of WPK1-5 and WPK5-mut provided by the present invention are:

Application of the polypeptide in the preparation of drugs with antithrombotic or anticoagulant activity:

The polypeptide WPK5 provided by the present invention has inhibitory activity on blood coagulation factor XIa after testing, and the IC ₅₀ is 978.20±52.15nM. The polypeptide WPK5-mut provided by the present invention has strong inhibitory activity on blood coagulation factor XIa after testing, and the IC ₅₀ is 8.34±0.20nM.

The polypeptide WPK5-mut provided by the present invention can prolong the time of ferric chloride-induced carotid artery thrombosis in a dose-dependent manner after testing, and the effective dose is 40 μmol/kg, and the effective dose of PN2KPI is 80 μmol/kg.

The polypeptide WPK5-mut provided by the present invention has no bleeding risk at the doses of 40 μmol/kg and 80 μmol/kg, which proves its safety.

Beneficial effect:

1. The present invention screens the Kunitz-type polypeptide sequence from the Whitmania Pigra salivary gland transcriptome database, constructs, expresses, screens, and replaces amino acids to obtain a series of polypeptides named WPK1-5, in which all The polypeptide sequence of the invention is a brand-new sequence. Although some of the sequences have low anticoagulant activity, they can still be used as prodrugs of polypeptide anticoagulant drugs, which can be modified. In addition, two loop amino acids were replaced on the basis of WPK5, and it was cloned and expressed as WPK5-mut.

In vitro drug efficacy experiments confirmed:

(1) WPK5 has inhibitory activity on coagulation factor XIa, IC ₅₀ is 978.20±52.15nM; WPK5-mut has stronger inhibitory activity on coagulation factor XIa, IC ₅₀ is 8.34±0.20nM.

(2) The polypeptide WPK5-mut dose-dependently prolongs APTT (activated partial thromboplastin time), but has no effect on PT (prothrombin time).

Experiments in animals have confirmed:

(1) The polypeptide WPK5-mut can dose-dependently prolong the time of ferric chloride-induced carotid artery thrombosis in mice, and its activity is better than that of PN2KPI.

(2) The polypeptide WPK5-mut has no obvious risk of bleeding at the doses of 40 μmol/kg and 80 μmol/kg in the mouse tail docking bleeding test.

In conclusion, the polypeptide WPK5-mut of the present invention has an anticoagulant effect, and its activity in vivo is stronger than that of PN2KPI. At the same time, it has no risk of bleeding under a certain dose, and can be used for the prevention and treatment of diseases such as brain injury (such as apoplexy, cerebral apoplexy). It is safe and effective, and is suitable for the research and development of drugs with antithrombotic or anticoagulant activity.

Description of drawings

The following drawings are used to illustrate specific embodiments of the present invention, but not to limit the scope of the present invention defined by the claims.

Fig. 1 is the electrophoresis diagram of WPK1-5 after purification;

Figure 2 is the electrophoresis diagram of WPK5-mut after purification;

Fig. 3 is the IC ₅₀ figure of WPK5 to coagulation factor XIa, a is the absorption value of 405nm of different concentrations; b is the inhibition rate;

Fig. 4 is the IC ₅₀ graph of WPK5-mut on blood coagulation factor XIa, a is the absorbance at 405 nm of different concentrations; b is the inhibition rate.

Detailed description of the invention

Unless otherwise defined herein, all scientific and technical terms used herein shall have the meaning commonly understood by one of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. In general, nomenclature and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not restrictive. Other features and advantages of the present disclosure will be apparent from the following detailed description and claims.

For a better understanding of the present invention, definitions and explanations of related terms are provided below.

As used herein, the term "about" when used in conjunction with a numerical value is meant to encompass a numerical value within a range having a lower limit of 5% less and an upper limit of 5% greater than the indicated numerical value.

The terms "polypeptide" and "peptide" as used herein refer to a polymeric form of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and modified The peptide backbone of the polypeptide. Polypeptides of the disclosure can be obtained by methods known in the art. Exemplary methods of making the polypeptides of the disclosure are set forth herein.

Polypeptides of the disclosure can be isolated and/or purified. The term "isolated" as used herein means has been removed from its natural environment. The term "purified" as used herein means increased purity, where "purity" is a relative term and not necessarily to be interpreted as absolute purity. In exemplary embodiments, the purity of the polypeptide (e.g., in a composition) is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, or At least about 98% or about 100%.

Polypeptides of the present disclosure may include one or more modifications including, but not limited to, phosphorylation, glycosylation, hydroxylation, esterification, cyclization, sulfonation, amidation, acetylation, carboxylation, lipid (e.g., myristoylated, palmitoylated), pegylated, albuminated, and other polypeptide modifications known in the art; or combined with one or more polypeptides that enhance stability, activity, and/or extend half-life The group or part of the conjugate. Modifications can increase the stability, activity and/or half-life of the peptide. For example, the C-terminus can be modified by amidation, addition of esters, addition of p-nitroaniline and thioesters. Can be modified by pegylation, acylation (e.g., lipopeptides), biotinylation, phosphorylation, sulfation, glycosylation, introduction of maleimide groups, chelating moieties, chromophores, and fluorophores N-terminus and side chain. The terms "half-life-extending moiety" and "half-life-extending group" are used interchangeably herein and are understood to refer to one or more chemical groups attached to one or more amino acid site chain functionalities , such as -SH, -OH, -COOH, -CONH2, -NH2, or one or more N and/or O glycan structures, and when conjugated to these proteins/peptides, can increase the protein/peptide in vivo Circulation half-life. Examples of half-life extending moieties include: biocompatible fatty acids and their derivatives, hydroxyalkyl starches (HAS) such as hydroxyethyl starch (HES), polyethylene glycol (PEG), poly(Glyx-Sery)n( HAP), hyaluronic acid (HA), heparin precursor polymers (Heparosan polymers; HEP), phosphorylcholine-based polymers (PC polymers), Fleximers, dextran, polysialic acid (PSA), Fc domains, transferrin, albumin, elastin-like peptide (ELP), XTEN polymer, PAS polymer, PA polymer, albumin binding peptide, CTP peptide, FcRn binding peptide, and any combination thereof.

The term "amino acid" as used herein refers to natural amino acids and unnatural amino acids, and amino acid substitutions (including from natural to unnatural) can be routinely performed by those skilled in the art while maintaining the function or efficacy of the original peptide.

Polypeptides of the present disclosure may comprise one or more (e.g., 1-10, 1-5, 1-3, 1, 2, 3, 4 or 5) amino acid substitutions, preferably conservative substitutions . The term "conservative substitution" as used herein refers to amino acid substitutions which do not adversely affect or change the essential properties of the protein/polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions in which an amino acid residue is replaced by another amino acid residue having a similar side chain, such as a physically or functionally similar residue (e.g., having similar size, shape, charge, chemical properties including formation of covalent bonds or hydrogen bonding ability, etc.) to the corresponding amino acid residue. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), with uncharged polarity Amino acids with side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with non-polar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g. threonine, valine, isoleucine ) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Accordingly, the corresponding amino acid residue is preferably substituted by another amino acid residue from the same side chain family. Methods for identifying amino acid conservative substitutions are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999 ); and Burks et al., Proc.Natl.Acad.Sci.USA 94:412-417 (1997), which are incorporated herein by reference).

"Percent (%) identity" with respect to a polypeptide is defined as the percentage of amino acids that are identical between a candidate polypeptide sequence and a polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Sequence alignments to determine percent identity between two amino acid sequences can be performed using various methods known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNASTAR) software . Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. Where percentages of sequence identity are referred to in this application, these percentages are calculated relative to the full length of the longer sequence, unless otherwise specifically indicated. Full-length calculations relative to longer sequences apply to both nucleic acid and polypeptide sequences.

The term "nucleic acid" as used herein refers to a polymeric form of nucleotides (ribonucleotides or deoxynucleotides) of any length. Thus, the term includes, but is not limited to, single-stranded, double-stranded DNA or RNA, genomic DNA, cDNA or nucleosides containing purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural or derived nucleosides Acid-base polymers.

The term "expression cassette" or "vector" as used herein refers to a recombinant nucleic acid, particularly recombinant DNA, which is used to express one or more specific nucleotide sequences, or to construct other recombinant nucleotide sequences.

A subject treated with a polypeptide or pharmaceutical composition of the present disclosure may have any of the diseases and/or symptoms described herein. The term "treating" as used herein refers to implementing a regimen to alleviate the signs or symptoms of a disease, which regimen may include administering one or more drugs to a patient. Desirable effects of treatment include decreased rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. Remission can occur before or after the appearance of signs or symptoms of a disease or condition. Thus, "treating" can include "preventing" or "preventing" a disease or undesired condition. Additionally, "treatment" does not require complete relief of signs or symptoms, does not require a cure, and specifically includes regimens that have only minor effects on the patient.

As used herein, the terms "effective amount" and "therapeutically effective amount" refer to the amount or dose of the polypeptide of the present invention which, after being administered to a patient in single or multiple doses, produces the desired effect in a treated subject, which includes Improvement of a subject's condition (eg, improvement of one or more symptoms) and/or delay in progression of symptoms, etc.

The terms "subject" and "patient" as used herein refer to humans or non-humans such as primates, mammals and vertebrates. In specific embodiments, the subject is a human.

The pharmaceutical composition used in the present invention comprises the aforementioned polypeptide or polypeptide mixture as an active ingredient, and one or more pharmaceutically acceptable carriers. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic or other adverse reactions when properly administered to a subject. The preparation of pharmaceutical compositions comprising polypeptides or additional active ingredients will be known to those skilled in the art in light of this disclosure.

As used herein, "pharmaceutically acceptable carrier" includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc.), non- Aqueous solvents (for example, propylene glycol, polyethylene glycol, vegetable oils and injectable organic esters, such as ethyl oleate), dispersion media, surfactants, antioxidants, preservatives (for example, antibacterial or antifungal agents, antibacterial oxidizing agents, chelating agents and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants and nutritional supplements, like these materials and their combinations. The pH and exact concentrations of the various components in the pharmaceutical composition are adjusted according to well known parameters.

The pharmaceutical compositions of the present invention may be administered in vivo to a subject in need thereof by various routes including, but not limited to, oral, intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, Intracardiac, intraventricular, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous and intrathecal, either by implant or inhalation. The compositions of the present invention can be formulated into preparations in solid, semi-solid, liquid or gaseous form; including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants and aerosols. Appropriate formulations and routes of administration can be selected according to the intended application and treatment regimen.

Those skilled in the art will appreciate that appropriate dosages may vary from patient to patient. Determining the optimal dosage generally involves balancing the level of therapeutic benefit against any risks or deleterious side effects. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the mode of administration, the time of administration, the bioavailability characteristics of the formulation to be administered; the chosen administration regimen; the rate of compound clearance, duration of treatment, any concomitant Therapy used, severity of condition, and species, patient's sex, age, weight, condition, general health, and previous medical history. The amount and route of administration of the compound are ultimately at the discretion of the physician, veterinarian or clinician, but dosages are generally selected to achieve local concentrations at the site of action that achieve the desired effect without causing substantial deleterious or adverse side effects.

Detailed ways:

The present disclosure provides anticoagulant Factor XIa polypeptides, nucleic acids encoding anticoagulant Factor XIa polypeptides, expression cassettes/vectors comprising said nucleic acids, and cells comprising any of the foregoing.

In particular, the present disclosure provides anti-coagulant Factor XIa polypeptides (referred to herein as WPK1, WPK2, WPK3, WPK4, WPK5).

The present disclosure also provides an anticoagulant Factor XIa polypeptide mutant WPK5-mut, wherein Loop1 (11TGPCRSNLER20) and Loop2 (34QYGGC38) in WPK5-mut are replaced by Loop1 (11TGPCRAMISR20) and Loop2 (34FYGGC38) in PN2KPI.

In some embodiments, the anticoagulant Factor XIa polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO:6.

In some embodiments, the anticoagulant Factor XIa polypeptide comprises 85% or more sequence identity (e.g., 90% or more, 95% or more, 97% or more) to an amino acid sequence selected from , 98% or higher, 99% or higher, or 100% sequence identity) amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO:6.

In some embodiments, the anticoagulant Factor XIa polypeptide comprises one or more (eg, 1-10, 1-5, 1-3, 1, 2, 3, Amino acid sequences with additions, deletions and/or substitutions of 4 or 5) amino acids: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6:

In some embodiments, the anticoagulant Factor XIa polypeptide is an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.

Various modifications and/or additions may be made to the polypeptides of the invention, including, for example, addition of additional amino acids or linkers to the sequence, without departing from the scope of the invention.

In some aspects, the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding an anti-Factor XIa polypeptide as disclosed herein, or a mutant thereof.

In some aspects, the invention relates to an expression cassette or vector comprising a nucleic acid molecule encoding an anti-Factor XIa polypeptide as disclosed herein, or a mutant thereof.

In some aspects, the invention relates to host cells comprising an expression cassette or vector as disclosed herein.

In some embodiments, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

In some aspects, the invention relates to a pharmaceutical composition comprising an anti-coagulant Factor XIa polypeptide or a mutant thereof as disclosed herein, and a pharmaceutically acceptable carrier.

In some aspects, the invention relates to the use of an anti-coagulant Factor XIa polypeptide as disclosed herein for the manufacture of a medicament with antithrombotic or anticoagulant activity.

In some aspects, the present invention relates to the use of an anticoagulant Factor XIa polypeptide as disclosed herein in the manufacture of a medicament for the treatment or prevention of diseases associated with thrombosis, including but not limited to stroke, stroke, acute Coronary artery syndrome, myocardial infarction, unstable angina, transient cerebral ischemia, and atherosclerosis; it is used in the preparation for the treatment and prevention of deep vein thrombosis and pulmonary embolism, and reduces the recurrence of acute deep vein thrombosis and pulmonary embolism Risky drug use. Prevention of venous thromboembolic disease (prevention of thrombosis in veins) is particularly concerned with thrombosis associated with orthopedic or general surgery. Treatment involved established deep vein thrombosis, with or without pulmonary embolism, with mild clinical symptoms, excluding pulmonary embolism requiring surgery or thrombolytic therapy.

In some aspects, the present invention relates to the use of the anticoagulant factor XIa polypeptide disclosed herein in the preparation of a medicament for treating or preventing cerebral apoplexy or apoplexy.

In some aspects, the invention relates to a method of treating or preventing a disease associated with thrombosis in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-coagulant Factor XIa polypeptide as disclosed herein or a mutant thereof or a pharmaceutical composition comprising an anticoagulant Factor XIa polypeptide or a mutant thereof as disclosed herein.

In some embodiments, the subject is a human or mammal suffering from a disease associated with thrombosis. In some embodiments, the subject or individual is a mammal, eg, a mouse or a human, preferably a human. The subject may suffer from, but not limited to, the following diseases: stroke, stroke, acute coronary syndrome, myocardial infarction, unstable angina, transient cerebral ischemia, atherosclerosis, deep vein thrombosis and pulmonary embolism.

In some embodiments, a polypeptide or pharmaceutical composition of the disclosure is administered alone. In alternative embodiments, a polypeptide or pharmaceutical composition of the present disclosure is administered in combination with another therapeutic agent, including but not limited to antiplatelet drugs, thrombolytic drugs, or other anticoagulant drugs, and the like. The antiplatelet drugs are selected from thromboxane A2 (TXA2) inhibitors (for example, aspirin), adenosine diphosphate (ADP) P2Y12 receptor antagonists (for example, clopidogrel, ticagrelor), platelet glycoprotein IIb /IIIa receptor antagonists (GPIs) (eg, tirofiban), phosphodiesterase inhibitors (eg, cilostazol), thromboxane synthase inhibitors (eg, ozagrel), platelet adenosine One or more of a cyclase stimulator (eg, prostacyclin) or a serotonin receptor antagonist (eg, sagrelate). The thrombolytic drug is selected from the first generation thrombolytic drug, the second generation thrombolytic drug or the third generation thrombolytic drug and the like. The first-generation thrombolytic drugs, for example, can be streptokinase and urokinase, which are extracts from bacteria, tissues or urine. The second-generation thrombolytic drug can be, for example, alteplase. The third-generation thrombolytic drug, for example, can be reteplase. Other anticoagulant drugs are selected from parenteral anticoagulant drugs, traditional oral anticoagulant drugs, new oral anticoagulant drugs and the like. The non-oral anticoagulant drugs are, for example, selected from heparin and its derivatives, including heparin, low-molecular-weight heparin, enoxaparin sodium, fondaparinux sodium, etc. These drugs themselves have no inherent anticoagulant activity. Combines specifically with antithrombin to exert anticoagulant activity. A direct thrombin IIa inhibitor, for example selected from bivalirudin, desirudin or argatroban. The traditional oral anticoagulant drugs, such as vitamin K antagonists, mainly include dicoumarins (warfarin) and indenediones, and these drugs act on multiple targets. For example, the targets of warfarin include coagulation factors II, VII, IX, and X. The new oral anticoagulant drugs mainly include direct thrombin inhibitors (dabigatran etexilate) and direct factor Xa inhibitors (rivaroxaban, apixaban, edoxaban) and the like.

In exemplary embodiments, a polypeptide or pharmaceutical composition of the present disclosure is administered concurrently with another therapeutic agent, including but not limited to antiplatelet drugs, thrombolytic drugs, or other anticoagulant drugs, and the like. The types of antiplatelet, thrombolytic, or other anticoagulant drugs can be selected as indicated elsewhere herein.

In alternative embodiments, a polypeptide or pharmaceutical composition of the present disclosure is administered before or after another therapeutic agent including, but not limited to, an antiplatelet drug, a thrombolytic drug, or other anticoagulant drug wait. Specific classes of antiplatelet, thrombolytic, or other anticoagulant drugs can be selected as indicated elsewhere herein.

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that these embodiments are provided by way of illustration only. Modifications and substitutions to the described embodiments may be made by those skilled in the art without departing from the scope and spirit of the invention. Accordingly, the invention is not limited to the described embodiments, but has its full scope as defined by the appended claims.

Example

The invention generally described herein will be understood more readily by reference to the following examples, which are provided by way of illustration and not intended to be limiting of the invention.

Those skilled in the art should understand that unless otherwise noted, the reagents, plasmids, cells, etc. used in the following examples are all commercially available products.

PN2KPI is based on literature reports (Navaneetham D et al.Structural and mutational analyzes of the molecular interactions between the catalytic domain of factor XIa and the Kunitz protease inhibitor domain of protease nexin 2.[J].The Journal of biological chemistry, 2005, 280( 43)), a total of 57 amino acids, including Loop1 and Loop2, can efficiently inhibit the carotid artery thrombosis induced by coagulation factor XIa and ferric chloride, and construct and express it for subsequent research.

Example 1: Discovery of WPK series polypeptides

Take the wide-bodied leech salivary gland, use magnetic beads to enrich mRNA, fragment it, synthesize the first strand and the second strand of cDNA with random primers, use QIAQuickPCR kit to purify, recover the target fragment by agarose gel electrophoresis, and amplify by PCR. Complete library construction. Illumina Hiseq 4000 sequencing was used, and Trinity (version 2.4.0) was used for de novo analysis of transcriptome without reference transcriptome, Blast annotation and Pfam analysis, and 5 sequences were searched with "Kunitz" as the keyword, named WPK1-5. The sequence information is as follows:

Example 2: Inhibitory effect of WPK series polypeptides on blood coagulation factor XIa

Construction of recombinant plasmid pPIC9k/WPK: Cloning the WPK1-5 sequence into pPIC9k using seamless cloning (cloning site NotI), linearized with SacI and electroporated into Pichia pastoris GS115, positive transformant colonies were identified by PCR and sequencing , Pick positive transformant colonies into the growth medium BMGY, 28.5 ° C, 220r/min shaking culture, when the OD600 of the bacterial solution is around 4-6, centrifuge at 4000r/min for 5min, and transfer the bacteria into the expression medium BMMY 1% methanol was added for induction, and after 72 hours, the supernatant was collected by centrifugation and purified to obtain the WPK1-5 target protein (Fig. 1). To test the inhibitory activity of WPK series peptides on blood coagulation factor XIa, pipette 100 μL FXIa (1nM) and 50 μL WPK1-5 (10 μM) and mix evenly in a 96-well plate, incubate at 37°C for 1 hour, add 50 μL substrate FXIa chromogenic substrate S2366 , the final concentration was 0.25mM, and the detection was continued for 1h at a wavelength of 405nm, scanning once every minute. Negative control: 50 μL TBS-BSA buffer + 100 μL FXIa + 50 μL substrate, blank control: 50 μL sample + 100 μL TBS-BSA buffer + 50 μL substrate (all wells are set as duplicate wells). Draw the absorbance value-reaction time curve, and the slope of the curve is the speed V of the enzymatic reaction, the speed V ₀ of the negative reaction, the speed V _i of the sample reaction, and the inhibition rate=(V ₀ -V _i )/V ₀ ×100%.

Conclusion: WPK5 has the strongest inhibitory activity on coagulation factor XIa in this series of polypeptides.

Example 3: The polypeptide mutant WPK5-mut sequence provided by the present invention and its comparison with the PN2KPI sequence

PN2KPI extensively interacts with XIa catalytic domain through Loop1 ( ¹¹ TGPCRAMISR ²⁰ ) and Loop2 ( ³⁴ FYGGC ³⁸ ), and exhibits good inhibitory activity against coagulation factor XIa. In this example, in order to further improve the inhibitory activity of WPK5 on blood coagulation factor XIa, the method of loop replacement was adopted, based on the new Kunitz skeleton provided by WPK5, two loops of PN2KPI ( ¹¹ TGPCRAMISR ²⁰ , ³⁴ FYGGC ³⁸ ) were used to target WPK- 5 two loops ( ¹¹ TGPRSNLER ²⁰ , ³⁴ QYGGC ³⁸ ) are replaced. And the amino acid sequence analysis of WPK5-mut and PN2KPI showed that the similarity was 60.78%.

Example 4: Cloning, expression and purification of the polypeptide mutant WPK5-mut provided by the present invention

Construction of the recombinant plasmid pPIC9k/WPK5-mut: Cloning the WPK5-mut sequence into pPIC9k using seamless cloning (cloning site NotI), using SacI to linearize and then electrotransfer into Pichia pastoris GS115, positive colonies were identified by PCR and sequencing , Pick positive colonies into the growth medium BMGY, 28.5 ° C, 220r/min shaking culture, when the OD600 of the bacterial solution is around 4-6, centrifuge at 4000r/min for 5min, transfer the bacteria into the expression medium BMMY, The final concentration of 1% methanol was added for induction, and the supernatant was collected by centrifugation after 72 hours, and the target protein was obtained by purification. Tricine-SDS-PAGE electrophoresis analysis showed that the recombinant WPK5-mut protein was highly expressed, and the WPK5-mut protein was 6.5kDa, which was consistent with the size of the expected recombinant protein (Figure 2).

Example 5: The inhibitory effect of polypeptide WPK5 and mutant WPK5-mut provided by the present invention on blood coagulation factor XIa

The polypeptide WPK5 and polypeptide WPK5-mut described in this example were obtained through recombinant expression in Pichia pastoris and nickel column affinity chromatography. The inhibitory effect of the recombinant polypeptide on blood coagulation factor XIa was detected by a chromogenic substrate method.

Pipette 100 μL of FXIa (1 nM) and 50 μL of WPK5 (0-57500 nM) and WPK5-mut (0-100 nM) in a 96-well plate and mix evenly, incubate at 37°C for 1 hour, add 50 μL of substrate, FXIa chromogenic substrate is S2366, and finally The concentration is 0.25mM, and the detection is continued for 1h at a wavelength of 405nm, and the scan is performed once every minute. Negative control: 50 μL TBS-BSA buffer + 100 μL FXIa + 50 μL substrate, blank control: 50 μL sample + 100 μL TBS-BSA buffer + 50 μL substrate (all wells are set as duplicate wells). Draw the absorbance value-reaction time curve, and the slope of the curve is the speed V of the enzymatic reaction, the speed V ₀ of the negative reaction, the speed V _i of the sample reaction, and the inhibition rate=(V ₀ -V) _i /V ₀ ×100%. Data processing was performed using the software Graphpad Prism 6.0 (Fig. 3a,b; Fig. 4a,b).

Conclusion: The polypeptide WPK5 has inhibitory activity on coagulation factor XIa, with IC ₅₀ of 978.20±52.15nM; the polypeptide WPK5-mut has strong inhibitory activity on coagulation factor XIa, with IC ₅₀ of 8.34±0.20nM.

Example 6: Anticoagulant activity of the polypeptide WPK5-mut provided by the present invention

In this example, the anticoagulant activity of the polypeptide mutant (WPK5-mut) provided by the present invention was detected by activated partial thromboplastin time (APTT) and prothrombin time (PT) methods. The polypeptide (WPK5-mut) described in this example was obtained through recombinant expression in Pichia pastoris and nickel column affinity chromatography (see Example 4).

Determination of activated partial thromboplastin time (APTT): Mix 40 μL of PPP and 10 μL of sample solution (normal saline for the blank control group) in a test cup, and incubate at 37° C. for 3 minutes. Add 50 μL APTT reagent and continue to incubate at 37°C for 3 min. Put the test cup in the test area, add magnetic beads, and then add 50 μL of CaCl ₂ solution that has been preheated at 37°C for 5 minutes to start the reaction immediately. When the small magnetic beads in the test cup stop rotating, it means that the experiment is over, and the fibrin formation time on the hemagglutination meter is read. The ability of prolonging APTT is analyzed by calculating the multiple value of prolonging coagulation time of the recombinant polypeptide, and the calculation formula is: prolonging APTT multiple=APTT value of each concentration measured/APTT value of blank control group. WPK5-mut prolongs APTT in a dose-dependent manner, at doses of 7.5 μM, 10 μM or 15 μM, APTT was 1.6-fold, 1.7-fold or 2-fold that of the control group, respectively.

Determination of prothrombin time (PT): Mix 40 μL PPP and 10 μL sample solution in the test cup, and incubate at 37°C for 3 minutes. Put the test cup in the test area, add magnetic beads, then add 100 μL of PT reagent that has been preheated for 5 minutes, and start the reaction immediately. When the small magnetic beads in the test cup stop rotating, it means that the experiment is over, and the fibrin formation time on the hemagglutination meter is read.

Conclusion: The peptide WPK5-mut dose-dependently prolongs APTT and has no effect on PT.

Example 7: The inhibitory effect of the polypeptide WPK5-mut provided by the present invention on ferric chloride-induced carotid artery thrombosis in mice and its activity comparison with PN2KPI in vivo

Mice (C57BL/6J, male, 18-22 g) were anesthetized by intraperitoneal injection of 5% chloral hydrate. After successful anesthesia, the mice were fixed in a supine position on a heating pad (37°C) to maintain body temperature. A midline incision was made in the neck of the mouse, and the fascia and muscle were gradually separated to expose the carotid artery, and the carotid artery was bluntly separated about 5 mm. Place a rubber strip (4×10mm) of suitable width under the artery to separate it from the surrounding tissue, keep the carotid artery clean, and administer WPK5-mut (10μmol/kg, 20μmol/kg, 40μmol /kg), PN2KPI (20μmol/kg, 40μmol/kg, 80μmol/kg) or normal saline for 10min, place it under a laser speckle instrument (Moor FLPI-2Moor Instruments) to observe the carotid artery blood flow. A filter paper strip (1*2mm) of FeCl ₃ solution was placed on the carotid artery for 3 minutes and removed, and the carotid artery was washed with normal saline for 3 times, and the carotid blood flow was continuously observed and recorded for 30 minutes. mFLPI2MeasV2-0 software was used for observation, and moorFLPIReviewV50 software was used for data analysis.

Conclusion: The polypeptide WPK5-mut dose-dependently prolongs the time of ferric chloride-induced carotid artery thrombosis in mice, and the effective dose is 40 μmol/kg; PN2KPI dose-dependently prolongs the time of ferric chloride-induced carotid artery thrombosis Formation time, the effective dose is 80μmol/kg. The activity of the polypeptide in vivo is stronger than that of PN2KPI.

Example 8: Effect of the polypeptide WPK5-mut provided by the present invention on mouse tail-docking bleeding

Mice (C57BL/6J, male, 18-22g) were anesthetized by intraperitoneal injection of 5% chloral hydrate, and after 10 min of tail vein injection of WPK5-mut (40 μmol/kg and 80 μmol/kg) or normal saline, the mice were placed Put the tail vertically in the fixer, measure it with a ruler, and mark it at 3mm from the tail tip, then use surgical scissors to cut off the mouse tail mark, and immerse the tail tip in 37°C physiological saline. Cut off the tip of the tail and start counting for 20 minutes, and take another stopwatch to record the accumulated blood flow time after cutting the tip of the tail (stop timing at the same time when the blood flow stops, and continue timing when bleeding occurs again). If the bleeding time t is greater than 20 minutes, record it as 20 minutes, t is the bleeding time.

Conclusion: The polypeptide WPK5-mut has no obvious bleeding risk at the doses of 40 μmol/kg and 80 μmol/kg compared with the normal saline group after the mouse tail docking bleeding test.

Claims (15)

1. Peptides, of which:

   (i) the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6;

   (ii) the polypeptide comprises an amino acid sequence having 85% or more identity to an amino acid sequence selected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 , SEQ ID NO:5, SEQ ID NO:6;

   (iii) the polypeptide comprises one or more (eg, 1-10, 1-5, 1-3, 1, 2, 3, 4 or 5) amino acids compared to an amino acid sequence selected from Added, deleted and/or substituted amino acid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6.
2. The polypeptide according to claim 1, comprising one or more modifications including but not limited to phosphorylation, glycosylation, hydroxylation, esterification, cyclization, sulfonation, amidation, acetylation, Carboxylation, lipidation, pegylation, albumination, and other polypeptide modifications known in the art; or conjugation to one or more groups or moieties that increase the stability, activity, and/or half-life of the polypeptide.
3. A nucleic acid molecule comprising a nucleotide sequence encoding one or more polypeptides of claim 1 .
4. An expression cassette or vector comprising the nucleic acid molecule of claim 3.
5. A cell comprising the polypeptide of claim 1, the nucleic acid molecule of claim 3, or the expression cassette or vector of claim 4.
6. The cell according to claim 5, wherein said cell is a bacterial cell, a fungal cell or a mammalian cell.
7. A pharmaceutical composition comprising the polypeptide according to claim 1 and a pharmaceutically acceptable carrier.
8. The pharmaceutical composition according to claim 7, which can be formulated into solid, semi-solid, liquid or gaseous preparations, including but not limited to tablets, capsules, powders, granules, ointments, solutions, suppositories, Injections, inhalants and aerosols.
9. Use of the polypeptide according to claim 1 in the preparation of drugs with antithrombotic or anticoagulant activity.
10. Use of the polypeptide according to claim 1 in the preparation of a medicament for treating or preventing a disease associated with thrombosis, the disease being selected from stroke, apoplexy, acute coronary syndrome, myocardial infarction, and unstable angina , transient cerebral ischemia and atherosclerosis; use in the preparation of medicines for treating and preventing the formation of deep vein thrombosis and pulmonary embolism, and reducing the risk of recurrence and pulmonary embolism after acute deep vein thrombosis.
11. A method for treating or preventing a disease associated with thrombosis in a subject, the method comprising administering a therapeutically effective amount of the polypeptide of claim 1 or the pharmaceutical combination of claim 7 to a subject in need thing.
12. The method according to claim 11, wherein the disease is selected from the group consisting of stroke, stroke, acute coronary syndrome, myocardial infarction, unstable angina, transient ischemia, atherosclerosis, deep vein thrombosis, and pulmonary embolism.
13. The method of claim 11, wherein the polypeptide or pharmaceutical composition is administered to the subject by intravenous, intraarterial, subcutaneous, parenteral, intranasal, intramuscular, intracranial, intracardiac, cardiac Indoor, intratracheal, oral, rectal, intraperitoneal, intradermal, topical, percutaneous and intrathecal, either by implant or inhalation.
14. According to the method of claim 11, said polypeptide or pharmaceutical composition is administered simultaneously with another therapeutic agent, said another therapeutic agent including but not limited to antiplatelet drugs, thrombolytic drugs or other anticoagulant drugs.
15. The method according to claim 11, said polypeptide or pharmaceutical composition is administered before or after another therapeutic agent, said another therapeutic agent including but not limited to antiplatelet drugs, thrombolytic drugs or other anticoagulant drugs .

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