RU2369615C2 - Direct thrombin inhibitor, possessing antiproliferative action - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: substance of polypeptide nature with molecular weight 14350 Da, with N-end amino acid sequence, homologous phospholipase A2 of snake venom, and possessing properties of direct thrombin inhibitor of mixed type as well as antiproliferative action is separated of cobra venom Naja haje by three-stage liquid chromatography.

EFFECT: invention enables to produce selective direct thrombin inhibitor, possessing antiproliferative action.

1 tbl, 3 dwg, 10 ex

Description

The invention relates to the field of biochemistry, in particular to a direct thrombin inhibitor, and can be used both in laboratory diagnostics and for the production of drugs for the treatment of diseases associated with thrombin hyperactivation and / or hyperproliferation.

Thrombin (EC 3.4.21.5), a serine protease, a key enzyme in the blood coagulation system, which turns fibrinogen into fibrin, the basis of a blood clot, is involved in the regulation of many physiological and pathophysiological processes, such as blood coagulation and anticoagulation mechanisms, thrombosis and fibrinolysis, and regulation of vascular tone , and in the regulation of the processes of development of the body, as well as in the processes of inflammation, tissue repair, atherogenesis, carcinogenesis and the development of Alzheimer's disease.

Untimely activation of the blood coagulation system in the arterial bed can lead to sudden cardiac arrest, acute coronary syndromes (myocardial infarction, etc.), peripheral vascular thromboembolism and stroke; in the venous bed, this can lead to deep vein thrombosis and to pulmonary embolism. Hyperproduction of active thrombin is also a leading pathogenetic link in intravascular coagulation syndrome. Since both the external and internal blood coagulation pathways have a common final stage - thrombin activation, which determines both the formation of a fibrin clot and platelet thrombus, inhibition of thrombin appears to be very logical in the treatment or prevention of such conditions.

Antithrombin-acting drugs are classified as direct thrombin inhibitors (PITs) (acting directly on the thrombin molecule - hirudin and its recombinant derivatives, as well as argatroban, melagatran), indirect thrombin inhibitors (heparins, fondaparinux), inhibitors of thrombin generation (inhibitor) (inhibitor) a group of recombinant analogues of endogenous anticoagulants (antithrombin, activated protein C, heparin cofactor II). Existing data prove that PITs have, for a variety of reasons, an advantage over other thrombin inhibitors in the treatment of these conditions, as well as in cardiac surgery.

However, the use of developed effective PITs is often limited by their individual disadvantages. So, hirudin and its recombinant analogues have a short half-life, as well as side effects such as bleeding tendency and anaphylactic reactions; most of the disadvantages of hirudin are absent in Girulog-1, a synthetic analog of hirudin, however, it itself is easily hydrolyzed by thrombin. Melagatran (in the form of a prodrug of ximelagatran), a low molecular weight PIT, is a highly active thrombin inhibitor and is practically devoid of most of the disadvantages of other PITs, but it turned out to have a hepatotoxic effect [Schwienhorst A. Direct thrombin inhibitors - a survey of recent developments // Cell Mol. Life Sci. - 2006. - V.63. - P.2773-2791].

Despite the huge selection of low molecular weight coagulation system effectors, drugs and diagnostics based on proteins from snake venoms are widely used to fine-tune the biochemical characteristics of hemostasis. In particular, in normal laboratory practice, thrombin-like enzyme (TPF) Reptilase from rattlesnake poison Bothrops atrox is used to determine the presence of pathological inhibitors of fibrin polymerization. For the diagnosis of lupus coagulants, fractions of the viper poisons Russell Daboja russelli and taipan Oxyuranus scutellatus containing enzymes activating factor X and prothrombin, respectively, are used. To determine the activity of Protein C, the enzyme Protak is used from the venom of the moth, Agkistrodon contortrix [Panchenko EP, Dobrovolsky AB Thrombosis in cardiology. Development mechanisms and treatment options. - M.: Sport and Culture, 1999. - 464 p.]. Anticistrons, TPF from Agomistrodon halys halys, in particular Ancystron-H, are used to determine the plasma fibrinogen level in patients who received heparin therapy [Gornitskaya OV, Platonova TN, Volkov GL Enzyme snake venoms. // Ukr. Bioxim. Zhurn. - 2003. No. 75. Issue 3. - S. 22-32]. In addition, for a more detailed study of hemostasis, a number of other components of snake venom are used, especially enzymes and disintegrins [Marsh N., Williams V. Practical applications of snake venom toxins in haemostasis // Toxicon. - 2005. - V.45. - P.1171-1181]. The reason for the widespread use of snake venom proteins in coagulology is their high selectivity with respect to their targets in the blood coagulation system, while they, unlike natural protein effectors, are often not inactivated by natural plasma inhibitors, which is their additional advantage as biochemical tools.

The thrombin-like enzyme Ankrod from Calloselasma rodostoma, a highly specific snake venom protein, can be used not only as a biochemical tool, but also as a medicine [Samsa G.P., Matchar D.B., Williams G.R., Levy D.E. Costeffectiveness of ancrod treatment of acute ischaemic stroke: results from the Stroke Treatment with Ancrod Trial (STAT) // J. Eval. Clin. Pract. - 2002. - V.8. - P.61-70].

Known coagulopathic phospholipases A2 contained in the venoms of snakes [Kini R.M. Structure-function relationships and mechanism of anticoagulant phospholipase A2 enzymes from snake venoms // Toxicon. - 2005. - V.45. - P.1147-1161]. Some A2 phospholipases are able to stop the proliferation of tumor cells [Makarova Ya. V., Osipov AV, Tsetlin VI, Utkin Yu.N. The effect of phospholipases A2 from snake venoms on the growth of neurites and the survival of the PC 12 pheochromocytoma cell line. // Biochemistry. - 2006. No. 71. - S.838-846], however, none of the known phospholipases is a thrombin inhibitor.

Known closest to the claimed thrombin inhibitor botrojaracin (Bothrojaracin). This is a preparation of a polypeptide nature isolated from rattlesnake venom; functionally, it is a mixed-type PIT (inhibition constant 15 nM). According to structural characteristics (molecular weight, subunit composition, N-terminal amino acid sequence), it belongs to lectin-like C-type proteins [Zingali RB, Jandrot-Perrus M., Guillin M.-C., Bon C. Bothrojaracin, a new thrombin inhibitor isolated from Bothrops jararaca venom: characterization and mechanism of thrombin inhibition. // Biochemistry. - 1993 .-- V.32. - P.10794-10802]; he has no differentiating properties.

The mentioned direct thrombin inhibitors do not completely satisfy all the requirements for such substances, therefore, there is a need to develop and obtain other thrombin inhibitors (including PIT) having a different mechanism of action and / or higher selectivity. The relationship between oncogenesis and blood hypercoagulation dictates the need to search for substances that act simultaneously on both of these processes.

The invention solves the problem of expanding the range of direct thrombin inhibitors.

The problem is solved by a new compound isolated by three-stage liquid chromatography from the Egyptian cobra venom Naja haje, consisting of one polypeptide chain, with a molecular weight of 14350 ± 14 Da, having seven intramolecular disulfide bonds, N-terminal amino acid sequence NVYQXRKMLG C AMPNG X-Y or W, and having an antiproliferative effect.

The first isolated new substance specifically inhibits thrombin and at the same time causes the differentiation of tumor cells.

The technical result is to obtain a direct thrombin inhibitor with antiproliferative effect.

It was found that a direct thrombin inhibitor isolated from Naja haje cobra venom has the N-terminal amino acid sequence NVYQYRKMLQ C AMPNGGPF, where X-Y or W, in addition to the direct inhibitory effect on thrombin, also has a specific one, i.e. unrelated to the fact of inhibition of thrombin antiproliferative effect. This substance expands the range of both biochemical anticoagulant reagents and possible drugs.

The obtained direct thrombin inhibitor can be used as a biochemical agent in the study of thrombosis, platelet aggregation, coagulation, tumor cell proliferation, especially for the selective study of the functions of thrombin in the hemostasis system, or for the selective suppression of thrombin activity in the study of other components of the blood coagulation system. Diseases that can be treated or studied using the direct thrombin inhibitor of the present invention are: thrombosis, atherosclerosis, restenosis, hypertension, angina pectoris, arrhythmia, heart failure, myocardial infarction, glomerulonephritis, thromboembolism, peripheral vascular disease, other cardiovascular diseases cerebrovascular accidents, inflammatory disorders, malignant neoplasms and other neoplastic diseases, as well as other conditions in which thrombin activation (and its rec torus) play a pathological role.

The direct thrombin inhibitor according to the invention consists of one polypeptide chain having a molecular weight of 14350 ± 14 Da according to MALDI mass spectrometry, and seven intramolecular disulfide bonds. Such a set of structural properties (polypeptide nature, molecular weight falling within the range of 13-15 kDa, N-terminal amino acid sequence of a single polypeptide chain, homologous to the above, 7 intramolecular disulfide bonds) is characteristic only of group I phospholipases A2 (EC 3.1.1.4).

The direct thrombin inhibitor according to the invention is obtained by three-stage liquid chromatography of the Egyptian cobra venom Naja haje (Fig. 1). The first stage is gel filtration using a carrier with such a pore size that allows you to separate the poison fraction with 13-15 kDa compounds from both the main toxic poison fraction (6-8 kDa) and high molecular weight proteins. Such a carrier may be Sephadex G-50 superfine resin (Amersham Biosciences) (Example 1). The second stage is cation exchange chromatography in a smooth gradient of salt concentration using a carrier with carboxymethyl active groups, whereby the direct thrombin inhibitor according to the invention is separated from both acidic and more basic components of the fraction, for example, on a Hema Bio CM HPLC column ("Tessek" ) (example 2). The third stage is reverse phase HPLC on a column with exposed hydrophobic groups, for example, with C 18 support, for final purification from components with close values of the isoelectric point and molecular weight, but having a different degree of hydrophobicity (Example 3).

Example 1. Carrying out gel filtration

800 mg of dried Naja haje cobra venom was dissolved in 1.5 ml of deionized water and applied to a gel filtration column (4.5 x 150 cm, Sepadex G-50 superfine, Amersham Biosciences, Sweden) in 0.1 M ammonium acetate buffer, pH 6.2, at a flow rate of 60 ml / h. The eluate is collected in test tubes in fractions of 15 ml, the optical density of the fractions is detected at a wavelength of 230 nm.

The collected eluate is combined, as shown in FIG. 1, A, concentrated and desalted by ultrafiltration on filters of the VivaScience system (polysulfone filters Vivaspin20, Sartorius AG, Germany) and lyophilized.

Example 2. Carrying out ion-exchange chromatography

The fraction V obtained after gel filtration was applied to an ion-exchange column (8 × 250 mm, carboxymethyl cellulose, NEMA-BIO, 1000 CM 10µm, Tessek, Czech Republic). Separation is carried out in a 5 mM Tris-HCl buffer, pH 7.5 in a NaCl gradient from 0 to 1 M per 100 min, at a flow rate of 1.4 ml / min. Fractions are collected using a fraction collector or manually according to the detector at a wavelength of 280 nm. The resulting fractions are concentrated and desalted on filters of the VivaScience system (polysulfone filters VivaSpin2 and VivaSpin6).

Example 3. Conducting reverse phase chromatography

Obtained after ion exchange chromatography, fraction 5 (Fig. 1, B) was separated on a reverse phase column (4.6 × 250 mm, Jupiter 5u, C-18, 300m, Phenomenex, USA) in an acetonitrile gradient in water from 15 to 45% in 30 minutes in the presence of 0.1% trifluoroacetic acid at a flow rate of 1 ml / min. Fractions are collected using a fraction collector or manually according to the detector at a wavelength of 280 nm. Solvents are removed from the obtained fractions by lyophilization. The peak of the target substance is indicated by a bar in FIG. 1, B.

The presence of a direct thrombin inhibitor (PIT) according to the invention in one of the poison fractions at each stage of purification is established functionally after desalination (ultrafiltration) using a thrombin time test (pronounced prolongation is observed); other components present in the fractions during purification do not interfere with determination. The standard test procedure is described in Example 6.

The PIT thus isolated is homogeneous and has a mass of about 14350 ± 14 Yes, which is confirmed by the method of MALDI mass spectrometry with an allowable spread of 0.1% for a given range of molecular weights. It has the N-terminal amino acid sequence NVYQXRKMLQ C AMPNGGPF, where X-Y or W, established by N-terminal degradation by the Edman method on an automatic protein sequencer or manually. The polypetid nature of the rest of the PIT is confirmed by proteolytic cleavage (Example 4). The presence in the polypeptide chain of 14 cysteine residues forming 7 intramolecular disulfide bonds is confirmed by total reduction with a thiol-containing reagent followed by alkylation (Example 5). The basic nature of the polypeptide compound is confirmed by the fact that it was retained by chromatography on a cation exchange column under the conditions described in example 2 (Fig. 1, B).

Example 4. Carrying out proteolytic cleavage with trypsin

5 μg of the polypeptide compound with completely reduced and alkylated, as described in Example 5, cysteine residues are dissolved in 20 μl of 50 mM phosphate buffer, pH 8.0, trypsin solution is added until the enzyme / substrate ratio is 1:50 by mass, incubated for 3 hours at 37 ° C. After desalting, the mixture is applied to the target to remove the MALDI mass spectrum. The disappearance of the peak with the initial molecular mass and the appearance of several peaks with masses not exceeding 3 kDa are observed on the spectrum, which indicates proteolytic cleavage of the rest of the molecule.

Example 5. Total recovery and alkylation of cysteine residues

200 μg of the obtained substance is dissolved in 200 μl of 50 mM phosphate buffer, pH 8.6, containing 6 M guanidine hydrochloride, 40 μl of a 6% solution of a reducing thiol-containing reagent (dithioerythritol or dithiotreitol, or mercaptoethanol, etc.) are added and incubated for 12 hours at room temperature. Then add 12 μl of 4-vinylpyridine and incubate for 3 hours at room temperature. The alkylated derivative, which is the main reaction product, is purified by reverse phase chromatography according to Example 3. The molecular weight of the derivative is 15820 ± 16 Da according to MALDI mass spectrometry, which indicates the unity of the polypeptide chain in the molecule and the inclusion of 14 pyridylethyl residues on 14 recovered cysteine residues. Carry out a similar experiment, but without the addition of a reducing reagent. The absence of changes in the molecular weight of the compound indicates the absence of free sulfhydryl groups in the starting material, i.e. all 14 cysteine residues form 7 intramolecular disulfide bonds in it.

In order to selectively suppress thrombin activity in in vitro experiments, the PIT according to the invention can be added to the blood plasma to a final concentration of 30-150 nm, while it does not affect other serine plasma proteinases.

The main functional feature of the PIT according to the invention is its ability to reliably inhibit the fibrinogenolytic activity of thrombin in blood plasma in concentrations above 7.5 nM, which is verified in the thrombin time test (Example 6), while it does not significantly affect concentrations up to 47.5 nM testimony of prothrombin time and APTT (the difference does not exceed 5% compared with control experiments) (examples 7 and 8), which demonstrates its selectivity for thrombin. When measuring the effect of the PIT according to the invention on the amidolytic activity of thrombin on a chromogenic substrate (Example 9), the direct nature of inhibition is confirmed and a predominantly non-competitive type of inhibition is established, which distinguishes it from most other PITs known in the art. Structurally, the PIT according to the invention is a protein representative of phospholipases A2, has an antiproliferative effect against tumor cells (in particular, rat pheochromocytoma cells) and anticoagulant properties.

The PIT according to the invention at a concentration of 15 nM prolongs the thrombin time of human blood plasma by 4 times, at a concentration of 30 nM - by 8 times, while the increase in prothrombin time and APTT does not exceed 5% compared with the test results in the absence of this substance (Fig. 2). The PIT of the invention inhibits the amidolytic activity of thrombin with respect to the synthetic chromogenic substrate N- (p-tosyl) -Gly-Pro-Arg-p-nitroanilide (Sigma-Aldrich) with an inhibition constant of about 70 nM. The fact of inhibition of thrombin in a system composed without other components of blood plasma, and the absence of increased inhibition with the addition of small amounts of plasma (0.8% or 3.3%) suggests a direct nature of the inhibition. The graphs of inhibition in the Lineuiver-Burke coordinates in the absence and in the presence of two concentrations of the substance converge at one point in the second quarter of the coordinates, which indicates a mixed type of inhibition (Figure 3). At concentrations above 150 nM (2 μg / L), the degree of inhibition of thrombin increases less dramatically (table). At a concentration of 1.5 μM and above, the indicated PIT significantly stops proliferation and causes the growth of neurite-like processes in tumor cells of rat pheochromocytoma of the PC 12 rat line (Example 10), and at the same time at concentrations of up to 15 μM it does not cause their death according to the standard MTT test ( tab.), i.e. does not exert a cytotoxic effect on them, which is a very important factor if applied in vivo. The cytotoxic properties of PITs are established on PC 12 cells using the MTT test according to the procedure described in the article [Makarova Y.V., Osipov A.V., Tsetlin V.I., Utkin Yu.N. The effect of phospholipases A2 from snake venoms on the growth of neurites and the survival of the PC 12 pheochromocytoma cell line. // Biochemistry. - 2006. No. 71. - S.838-846]: after 24-hour incubation with the test compound, the number of surviving cells is determined by staining with a 0.05% solution of 3- (4,5-dimethylthiazolyl-2) -2,5-diphenyl-2H-tetrazolium bromide (MTT) for 1.5 hours. The resulting crystals of formazan are dissolved in dimethyl sulfoxide, the optical density is measured on a flat spectrophotometer (Multiscan) at a wavelength of 540 nm and the readings are compared with the test results in the absence of the test compound. At a concentration of up to 15 μM, the PIT according to the invention does not affect the activation of the serum complement system in the classical way or at a concentration of 1.5 μM in an alternative way, which can be determined in standard hemolytic tests based on lysis of sheep erythrocytes sensitized by rabbit antibodies and guinea pig serum or rabbit erythrocytes with human serum, respectively, as described, for example, in [Shoibonov BB, Osipov AV, Kryukova EV, Zinchenko AA, Lakhtin VM, Tsetlin VI, Utkin Yu.N. Oxiagin from the Naja oxiana cobra venom is the first reprolysin inhibiting the classical pathway of complement. // Mol. Immunol. - 2005. - V.42. - P.1141-1153], which also indicates that the PIT according to the invention does not affect the remaining serine proteinases of blood plasma. Using the above methods, as well as the methods described in examples 6-10, establish the selectivity of PIT, the absence of cytotoxic and the presence of antiproliferative effects.

The compound, a direct thrombin inhibitor of the present invention, has anticoagulant activity as well as antiproliferative effect. It can be used as a biochemical agent in the study of thrombosis, platelet aggregation, coagulation, and proliferation of tumor cells. Thus, the selective, predominantly non-competitive nature of direct inhibition will allow a more selective study of the state of the general stage of the blood coagulation cascade carried out by active thrombin and the state of the remaining components of the cascade, for example, the contribution of an activator or an early stage inhibitor in the selective inhibition of thrombin by the compound of the invention. Moreover, changes in the activation of other stages of the cascade by the compound according to the invention as compared with the degree of prolongation of thrombin time (and their possible contribution to it) will be minimal (less than 5% at the concentrations indicated in examples 6-9).

Example 6. The effect on the test thrombin time

100 μl of normal citrate plasma is incubated for 5 min at 37 ° C with 5 μl of a 0.0005% aqueous solution of PIT according to the invention. Then, at room temperature, 20 μl (1U / ml) of human thrombin solution is added and the formation of a fibrin clot on the coagulometer or visually is noted. In a control experiment, instead of a solution of the compound of the invention, 5 μl of water is added. The increase in clot formation time is 800% compared with the control experiment. 100 μl of a 0.3% fibrinogen solution is incubated for 5 minutes at 37 ° C. with 5 μl of a 0.0005% aqueous solution of PIT according to the invention. Then, at room temperature, 20 μl (1U / ml) of human thrombin solution is added and the formation of a fibrin clot on the coagulometer or visually is noted. In a control experiment, instead of a solution of the compound of the invention, 5 μl of water is added. The increase in clot formation time is 800% compared with the control experiment.

Example 7. The effect on the test APTT

100 μl of normal nitrate plasma is incubated for 15 min at 37 ° C with 20 μl of a 0.0005% aqueous solution of PIT according to the invention, then 100 μl of APTT reagent based on ellagic acid is added and the clot formation time is noted. In a control experiment, instead of a solution of the compound of the invention, 20 μl of water is added. The increase in the time of clot formation is 0-5% compared with the control experiment.

Example 8. The effect on the test readings of prothrombin time

100 μl of normal citrate plasma is incubated for 5 min at 37 ° C with 20 μl of a 0.0005% aqueous solution of PIT according to the invention, then 100 μl of thromboplastin-based reagent is added, and incubation is continued for another 3 min at 37 ° C. The coagulation reaction is initiated by adding 100 μl of a 20 mM calcium chloride solution and the time of clot formation is noted. In the control experiment, instead of the PIT solution according to the invention, 20 μl of water is injected. The increase in the time of clot formation is 0-5% compared with the control experiment.

The results obtained in examples 7 and 8 indicate the selectivity of the PIT according to the invention for thrombin, since it does not affect the activation of the external and internal blood coagulation pathways and, therefore, other serine coagulation system proteases.

Example 9. Inhibition of amidolytic activity of thrombin

To 100 μl of an aqueous solution of the chromogenic synthetic substrate N-Tos-Gly-Pro-Arg-pNA (Sigma-Aldrich) in several dilutions (from 30 μM to 3 mm) in 10 mm imidazole buffer, pH 7.4, add 20 μl 0 , 15 μM and (in a parallel experiment) 0.3 μM PIT solution according to the invention, the reaction is induced by the addition of 20 μl of 0.5 U / ml thrombin. The optical density of the reaction mixture is detected at a wavelength of 405 nm every 30 seconds, until it reaches a plateau. In the control experiment, 20 μl of water is injected instead of the PIT according to the invention. Three graphs are constructed (corresponding to two inhibitor concentrations and control) of the dependence of the reaction rate on the concentration of the substrate in the inverse coordinates of the Lineuiver-Burke. The constant and type of inhibition are determined based on the values of the slope angles and the location of the intersection point of the graphs, respectively.

Since tumor progress and activation of the coagulation system are interconnected, the PIT of the present invention can be used to conduct experiments to study the effect of simultaneous suppression of both processes - tumor cell proliferation and hypercoagulation.

Example 10. The influence of the PIT according to the invention on the differentiation of tumor cells

Rat PC 12 pheochromocytoma cells were cultured at 37 ° C. and 5% CO ₂ in RPMI 1640 medium (Sigma) containing 15% fetal calf serum (Hyclone) and 2 mM glutamine. To study the effect of phospholipases, cells are seeded in a 96-well plate (Corning & Costar) with a density of 5-10 · 10 ⁴ cells per well (60-70% monolayer) and incubated in medium with 1% serum for 24 hours. Then add the PIT according to the invention in various concentrations and the incubation is continued under the same conditions. After 24 hours, the morphological changes observed with the growth of neurites are detected visually through microscopy and recorded in photographs. Differentiated are cells in which the length of the neurites exceeds the length of the cell body. For the quantitative determination of neuritogenic activity, the percentage of differentiated cells is calculated in relation to the total number of visible cells (table).

In addition, the PIT of the invention can be used to produce a pharmaceutical composition comprising a therapeutically effective amount of a direct thrombin inhibitor in a pharmaceutically acceptable carrier, for example, lyophilized powder for injection in aseptically sealed ampoules. The drug is readily soluble in water for injection.

Given the effectiveness of the PIT according to the invention in vitro (at a concentration of 30 nM increases the thrombin time for whole plasma by 8 times) and the volume of circulating blood in the body, the average single dose can be considered 33-40 μg / kg body weight, and the daily dose for treating the disease or the conditions indicated above are from about 0.01 to 1 mg / kg body weight per day. Thus, for an average body weight of 70 kg, the dosage level is from about 0.7 to 70 mg of the drug per day, in a single dose or 2-5 fractional doses.

Thus, the invention extends the range of PIT. Compared to the closest analogue, botrojaracin, the claimed PIT is similar to it in terms of source of excretion (snake venom), chemical type (polypeptide compound), and anticoagulant activity (direct mixed-type thrombin inhibitor). The direct thrombin inhibitor according to the invention differs structurally from botrojaracin in that it belongs to a different class of proteins (phospholipases A2), and functionally in that it is able, independently of its thrombin-inhibiting activity, to stop the proliferation of tumor cells, causing their differentiation.

Claims (1)

A direct thrombin inhibitor isolated by three-step liquid chromatography from Egyptian cobra venom Naja haje, consisting of one polypeptide chain with a molecular weight of (14350 ± 14) Yes, determined by MALDI mass spectrometry, having seven intramolecular disulfide bonds, an N-terminal amino acid sequence NVYQXRKMLQ C AMPNGGPF, where X is Y or W, and having anti-proliferative effect.

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