A method of obtaining a polypeptide, capable of blocking the calcium channel

 

(57) Abstract:

Usage: for the treatment of diseases caused by disruption of the functioning of calcium channels. The inventive polypeptide isolated from the venom of the spider Agelenopsis aperta chromatography in the range of 20 to 230 nm on a column of type C - 18 22 250 mm, particle size 10 μm. Column elute with a mixture of acetonitrile and 0.1% aqueous triperoxonane acid in a gradient increasing Stephania acetonitrile according to the program: 0 - 30 min 5 - 20% content of acetonitrile, 30 - 35 min - 20 - 70% content of acetonitrile in 0.1% triperoxonane acid. The flow rate is 15 ml/min 41.5 minute collect fraction and subjected her to chromatography on a column of the same type 10 250 mm, particle size 5 µm. Column elute at a flow rate of 3.5 ml/min by isocratic solvent system of 70% of 0.1% aqueous triperoxonane acid and 30% acetonitrile in the same wavelength range. Collect the fraction of 7.5 minutes, put it lyophilization. Get polypeptide mol. m 5201. Define aminobenzene amino acid sequence.

The invention relates to a polypeptide found in the venom of the spider Agelenopsis aperta, and to polypeptides having essentially the same amine and their acceptable from a pharmaceutical standpoint salt block calcium channels in cells, including neural and muscle cells of various organisms, including invertebrates and vertebrates. The proposed method also applies to the use of these polypeptides and their salts to block calcium channels in cells, such as cells of the nervous and muscular systems of the body, in the treatment of diseases and symptoms caused by disruption of the functioning of calcium channels in a mammal, and for combating invertebrate pests. In addition, the invention relates to compositions containing these polypeptides and their salts.

It is known that the venom of the spider Agelenopsis aperta contains at least two of the toxin, which have an impact on calcium fluxes (Jackson, etc. So. Heu. Sci.Astr, T. 12, 1987 S. 1078). The toxin has a molecular weight less than 1000 daltons and slows calcium flows in a variety of tissues.

In another paper (Jackson. , And other Soc. Heu.Sci.Astr., so 12, 1986, S. 730) described another toxin from Agelenopsis aperta containing component at about 6000 meters century This toxin performs presinapticescuu blockade signal and blocks the calcium channels associated with the release of a neurotransmitter.

Some polypeptides that are contained in the venom of the spider Agelenopsis aperta, known from the application of the U.S. N om in accordance with fractions in which they are found .

Fraction C. Aminobenzene amino acid sequence containing H2N-glu-lys-gly-leu-pro-glu-gly-ala-glu-cys-asp-gly-asn-glu-ser-asp-cys-lys-cys-ala - to-gly-gen-trp-ile-lys-cys-arg-cys-pro-trp-lys-trp--his-ile-thr-gly-glu-gly-pr o-cys-t-gly-leu-lys-lys-thr-cys-ile-ser-lys-leu-ser-asp-pro-asn-arg-asn-glu-trp-leu-ser-,

mol.wt. for the complete polypeptide in accordance with FAB MS:7267.

The fraction of N1.

H2N-ala-cys-val-gly-ghe-asn-gen-gem-cys - to-ala-asp-trp-ala-gly-prp-his-cys-cys-asp-gly- -tyr-tyr-cys-thr-cys-arg-tyr-phe-pro-lys-cys- -ile-cys-arg-asn.

The fraction of N2. Aminobenzene amino acid sequence containing H2N-ala-lys-ala-leu-pro-pro-gly-ser-val-cys- -asp-gly-asn-glu-ser-asp-asp-cys-lys-cys-tyr- -gly-lys-trp-his-lys-cys-ary-cys-pro-pro-lys-gly- -his-phe-thr-, m wt. full polypeptide in accordance with FAB M: 5494.

Fraction 1. H2N-asp-cys-val-gly-glu-ser--gen-gen-cys-ala-asp-trp-ala-gly-pro-his-cys- -cys-asp-gly-tyr-tyr-cys-thr-cys-arg-tyr-phe - pro-lys-cys-ile-cys-val-asn-asn-asn-CONH2; FAB MS; 4158.

Faction J. Aminobenzene amino acid sequence containing H2N-asp-glu-pro-cys-ile-pro-leu-gly-lys-ser-cys--ser-trp-lys-ile-gly-thr-pro-tyr-pro-asp-asp-ala-, m wt. for the complete polypeptide in accordance with FAB MS:5505.

Faction K. H2N-glu-asp-asn-cys-ile-ala- -glu-asp-tyr-gly-lys-cys-thr-trp-gly-gly-thr-lys-cys-cys-arg-gly-arg-pro-cys-arg-cys-ser-met-ile-gly-thr-asn-cys-glu-cys-thr-pro-arg-leu-ile-met-glu-gly-leu-ser-phe- ala - CONH2, FAB MS: 5274.

F. the y-asp-tyr-pro-trp-met-val-ser-ile-gen-gen-lys-asn-lys-lys-gly-gly-phe-asp, estimated M. wt. for the complete polypeptide is about 20000.

Fraction L2. The polypeptide having the following identifying characteristics:

(a) is contained in a fraction from the crude venom of the spider Agelenopsis aperta, which elute in the column Vidax-18, h mm, pore size 300 , particle size 10 µm, is used volumetric rate of 15 ml/min, solvent system with linear gradient: 5%->>20%, and 95%>>80% [0->>30 min], then 20% ->>70%, 80% ->>30% And [30->>55 min], where a is 0.1% aqueous TFA (triperoxonane acid); B - acetonitrile N2approximately 43 minutes;

b, is contained in the fraction that is described in and which elute through the column type video recorder C-18, h mm, pore size 300 , particle size 5 μm, is used, the volumetric rate of 3.5 ml/min. and a solvent system with a linear gradient: 25%->>40%, 75%->> ->>60% [0->>30 min.] where a is 0.1% of aqueous TFA; acetonitrile at approximately 22,5 minute.

Fraction M Aminobenzene amino acid sequence containing H2N-glu-ala-thr-glu-ala-ala-lys-val-len - ser-asn-leu-asp-glu-thr-val-asp-pro-approximate m wt. for a complete polypeptide of approximately 80000.

Compounds that are antagonists of calcium are sa the diseases, as angina, hypertension, cardiomyopathy, adventcalendar arrhythmia, ezofagealnaya achalasia, premature birth and disease, Raynaud's disease among others. (Nailer U. J. Calcium antagonists. New York: Academic Press, Harcourt Here Brace Javanovich Publishers, 1988). In addition, these compounds are used for studying the physiology of cells such as neural and muscle cells, and in combating invertebrate pests.

The invention relates also to a polypeptide found in the venom of the spider Agelenopsis aperta. The specified polypeptide, and the fraction in which it is present, in accordance with the present invention can be described as follows.

Fraction q

a) is contained in a fraction from the crude venom of the spider Agelenopsis aperta, which elute in the column of the video recorder C-18, h mm, pore size 300 , a particle size of 10 μm, is used, the volumetric rate of 15 ml/min, solvent system consisting of programs with linear gradient: 5%->>20% [0->>30 min] , then 20%->>70% [30->>55 min], where a is 0.1% aqueous triperoxonane acid (TFA) In acetonitrile at about 41,5 minute;

b) is contained in the fraction described in and which elute in the column of the video recorder C-18, h mm, pore size 300 , particle size 5 μm, is used volumetric CCM; In - acetonitrile within 7.5 minutes;

C) aminobenzene amino acid sequence containing

H2N - hys-lys-lys-cys-ile-ala-lys-asp-tyr- -gly-arg-cys-lys-trp-gly-gly-thr-pro-cys-cys- -arg-gly-arg-gly-cys-ile-cys-ser-ile-met-gly-thr- -asn-cys-glu-cys-lys-pro-arg-leu-ile-met-glu--gly-leu-.

Polypeptides that are the subject of the present invention, blocking the calcium channels in the cells. Therefore, these polypeptides are used to block calcium channels. The polypeptide used for combating invertebrate parasites and treating diseases and symptoms in mammals, related to the functioning of calcium channels in cells. In addition, the proposed polypeptides, have essentially the same amino acid sequence and substantially the same blocking the calcium channels activity, which is known polypeptide.

The present invention relates also to pharmaceutical compositions containing these polypeptides, and methods of using the polypeptides.

The poison is obtained from the spider Agelenopsis aperta through the process of "milking" using electrical stimulation in accordance with standard techniques. In the preferred embodiment, using this technique, which would guarantee the absence of impurities in Zeleny poison stored in a frozen state at a temperature of about -78aboutWith before using it to clean.

The cleaning component of the whole venom carried out using reversed-phase high-performance liquid chromatography (HPLC) on a variety of preparative and prepreparation columns, such as column type video recorder C-4 and C-18 (firm Rainin Tool Co.Inc., Mack Road, Woburn Massachusetts 01801). Detection of peaks exercise monochromatically in the range of 220-230 nm. Subsequent analysis of these fractions can be carried out, for example, using polychromatic UV data collected using a detector with a set of diodes type waters 990 (company Millipore Corporation, Warters Chromatography division, 34 maple Street, Milford, Massachusetts 01757). Fractions from the column are collected by means of known techniques, such as the use of collector fractions ISCO/"FOXY" and detector peaks ISCO 2159 (firm ISCO, 4700 superior, Lincoln, Nebraska 68504). These fractions are collected in vessels of appropriate size, for example a sterile plastic laboratory jars. Concentration of the fractions is then carried out using lyophilization of the eluent followed by lyophilization from water. The purity of the obtained fractions of the components can be determined using chromatographic anal the EMA, used for the final purification of the fraction.

Patterns contained in the respective fractions, determined in accordance with known analytical techniques such as mass spectroscopy and nuclear magnetic resonance. The sequence of the polypeptide fraction Q determined using known methods. For example, S-pyridylmethylamine cystine residues of the polypeptide may be carried out in solution with subsequent analysis of the amino acid sequence of the polypeptide. One such procedure for the S-pyridylmethylamine is the following. About 1-10 mg of the polypeptide is dissolved or diluted in approximately 50 ml of the buffer obtained by mixing parts 1M l, pH 8.5, containing 4 mm etc, and 3 parts of 8M guanidine, Hcl. Then add 2.5 ml of 10% aqueous solution of 2-mercaptoethanol and the mixture is incubated at room temperature in the dark under an argon atmosphere for 2 h After incubation add 2 ml of 4-vinylpyridine) - derivatives (fresh reagent stored in an argon atmosphere at -20aboutC) and the mixture incubated for 2 h at room temperature in the dark under argon. Here the mixture absoluut, in the preferred embodiment, using a short column for reversed-phase hrynyk techniques.

In the implementation of the proposed method, it was found that the corresponding column for the initial fractionation of the venom is a column of type video recorder C-18, h mm, pore size 300 , a particle size of 10 μm. This column elute with a bulk velocity of 15 ml/min using a linear gradient: 95%->>80% And 5%->>20% (0->>30 min), then 80%->>30% And 20%->>70% (30->>55 min), where a is 0.1% water triperoxonane acid (TFA); B - acetonitrile. These fractions are collected. Thus obtained fraction selected for further purification. The time of elution fraction is approximately of 41.5 minutes

Fraction Q1subjected to further purification using column type video recorder C-18, h mm, pore size 300 , particle size 5 µm, with a volume rate of 3.5 ml/min using isocratic system 70% of 0.1% aqueous TFA, 30% acetonitrile. These fractions are collected.

Fraction Q elute from the column for approximately 7.5 minutes Established that faction, which was suirable at approximately 8,3 minute, contains a polypeptide that is known as fraction A. Fraction Q contains a polypeptide comprising aminobenzene amino acid sequence containing

H2N-lys-lys-lys-cys-ile-ala-lys-asp-tyr- -gly-arg-cys-lys-trp-gly-gly-thr-pro-cys-cys- -arg-gly-arg-gly-cys-ile - cys in a fraction Q of the venom of the spider Agelenopsis aperta, it is possible to obtain the connection specified by the procedures of isolation/purification from whole venom. The polypeptide may be obtained using techniques of recombinant DNA through cloning the coding sequence for the polypeptide or its parts. For example, probes for hybridization, which have the advantage for the amino acid sequences of the specified polypeptide, can be used in accordance with well known techniques for cloning the coding sequence for the complete polypeptide. The combination of the techniques of recombinant DNA and protein synthesis in-vitro can also be used to obtain polypeptides. Such techniques of protein synthesis in-vitro include the use of synthesizer peptides in solid phase AS A (firm Applied Biosystem, Inc., Lincoln center Drive, 850, foster city, California 94404) using standard chemistry Merrifield or other chemistry in the solid phase.

It is known that certain amino acid substitutions can be made in the polypeptide, which do not violate or essentially do not disrupt the function of the polypeptide. Specific possible substitution vary from polypeptide to the polypeptide. Defined the holding essentially the same amino acid sequence and having the same activity by blockade of calcium channels, contained in the field covered by the present invention.

Polypeptides irreversibly blocking the calcium channels, contained in different cells, such as nerve and muscle cells of invertebrates and vertebrates.

The ability of the polypeptides to block calcium channels is confirmed by using the following procedure. The cerebellar mast cells derived from cerebellum region at the age of 8 days. Squares (1 cm2from Aclara cover poly-L-lysine and placed in a Cup with 12 cavities, which contain 1 ml of the basal medium IGLTA. Cells dissociate and portions containing 6,h6CL add in each recess containing the squares of Aclara. After this procedure, 24 h was added to the cytosine-beta-D-arabino furanoside (final concentration 10 mm). Cells use for pure analysis on 6, 7 and 8 days of cultivation. Cells adhering to the squares of Aclara, transferred into a Cup with 12 cavities containing 1 ml of 2 mm Fura/S (firm Molecular of Probes Inc., APEC, PRS, 97402) in the buffer S containing 0.01% bovine serum albumin, 0.01% of dextrose, pH 7.4; magnesium no. Cells incubated for 40 min at 37aboutS; buffer containing Fura/S removed and replaced with 1 ml of the same buffer but without fur/AM. In quartz the e is inserted in a temperature-controlled (37about(C) the holder is equipped with a magnetic stirrer; the fluorescence was measured by fluorescence spectrophotometer. The fluorescence signal allow to stabilize for about 2 minutes Later in the cuvette add 5-20 ml raw solution of the studied compounds in bateriafina phosphate salt solution (BFSR, pH 7.4) in appropriate concentrations. Calibration of the fluorescence signal and the correction due for/AM carried out using known procedures Nemeth and others (J. Biol.Chem, I. 262, 1987, S. 5188), upon completion of each test the maximum fluorescence (Fmax) is determined by adding ionomycin (35 mm) and the minimum fluorescence value (Fmindetermine by further adding EGTC (12 mm) in helically. Using this procedure, the blockade of calcium channel offered by the connection set by the decrease in fluorescence upon addition of the compounds.

The polypeptide of the present invention is used as blockers of calcium channels in cells. The proposed connection can also be used for combating invertebrate parasites and treating diseases and symptoms caused by impaired functioning the RNA arrhythmia, premature birth and disease Raynaud's syndrome. In addition, the proposed compounds are used in the study of physiology of cells, including cells of the nervous and muscular systems.

In addition, in the field covered by the present invention, are acceptable from a pharmaceutical point of view salts of polypeptides. Such salts are produced by procedures well known to every expert. For example, alkali metal salts of polypeptides can be obtained in accordance with known techniques.

If the polypeptide is intended for a mammal, then it can be used alone or in combination with a pharmaceutically acceptable carrier or diluent in a pharmaceutical composition in accordance with pharmaceutical practice. The polypeptides can be applied tematicheskie or parenteral and parenteral preferred method for these polypeptides. Parenteral administration includes intravenous, intramuscular, intraperitoneal, subcutaneous and local methods.

When staticheskom the use of the polypeptide compound can be applied, for example, in the form of tablets or capsules, or in the form of an aqueous solution or suspension. In the case of tablets for tematicheskoe COI is tearin magnesium. For tematicheskoe use in capsule form, useful diluents include lactose and dried corn starch. If tematicheskoe apply the appropriate suspension in water, the active ingredient is combined with emulsifying and suspendresume agents. If necessary, you can add flavors and/or flavoring agents.

For intramuscular, intraperitoneal, subcutaneous and intravenous use in General receive sterile solutions of the active ingredient and pH of the solutions should be suitably adjusted and bateriafina. For intravenous use, the total concentration of solutes should be adjusted to obtain isotone preparation.

If the polypeptide or its salt, which is the subject of the present invention, used for humans, the daily dose may be generally determined by the attending physician. In addition, the dose may vary depending on age and weight, as well as individual reactions and the severity of the patient's symptoms; the dose also depends on the activity of the specific compound.

If the polypeptide or its salt is used to control invertebrate parasites who should be sprayed in the form of a solution in an invertebrate. The number of connections required for combating invertebrate taxa varies depending on the type bespozvonochnykh and environmental conditions and can be determined by a specialist using this connection.

If the polypeptide or its salt is used for physiological studies of cells, the polypeptide used in accordance with techniques well known to every expert. For example, the polypeptide may be applied to cells in an appropriate physiological buffer. The concentration of the compounds for use in such studies is equal to 100 mm. However, the concentration of such a polypeptide may be higher or much lower than 100 mm. The number of used connections can be determined by any expert using well-known methods.

P R I m e R 1. Initial fractionation of whole venom of the Agelenopsis aperta.

Whole venom of the spider Agelenopsis aperta, received from the company Natural Product science Inc. , Salt lake city, Utah 841008, and which is kept in a frozen state at a temperature of about -78aboutC, thawed, and portions of it in 10-60 ml, diluted to 200 ml, was loaded onto the column type video recorder C-18 (h mm, pore size 300 , particle size 10 μm) and was suirable using surround with the t;70%, 80% ->>30% And [30->>55 min], where a is 0.1% aqueous TFA; B - acetonitrile. Detection of the peaks was carried out monochromatically in the range of 220-230 nm, and fractions were collected using a collector fractions ISCO/"FOXY" and detector peaks ISCO 2159. Fractions were collected from 20 to 60 minute. Fraction Q1collected on approximately 41,5 minute.

P R I m m e R 2. Subfractionated fraction Q1and defining structures.

Fraction Q1obtained in example 1, was loaded onto the column type video recorder C-18 (h mm, pore size 300 , particle size 5 μm) and suirable from it, using the volumetric rate of 3.5 ml/min and the isocratic solvent system of 70% A, 30% B, where a is 0.1% aqueous TFA; B - acetonitrile. Detection of the peaks was carried out using a detector with a series of diodes waters 990, fractions were collected as in example 1. Two fractions were obtained as follows: a Fraction of the Time of elution Q approximately 7.5 minute To about 8.3 minute

Fractions Q and K, which contained polypeptides, and then prepared for sequence analysis using lyophilization of the eluent followed by lyophilization from water in accordance with known techniques.

Analysis of the alkylated amino acids of the polypeptide of fractions Q and was carried out using Peak was collected using the analyzer sequence of the protein-peptide company Applied of Biosystems model 4705 with turning water TPA. Analysis of the resulting phenylthiohydantoin amino acids was performed in the "online" using the analyzer PTH company Applied of Biosystems model 120A or in the "off line" on the PTH column of the firm DuPont ZORBAX.

The analysis of amino acids was determined aminobenzene amino acid sequence of part of the polypeptide contained in the fraction Q, which has the following form:

H2N-lys-lys-lys-cys-ile-ala-lys-asp-tgr-gly-arg-cys-lys-trp-gly-gly-thr-pro-cys-cys- -arg-gly-arg-gly-cys-ile-cys-ser-ile-met-gly-thr- -asn-cys-gly-cys-lys-pro-arg-leu-ile-met-glu-gly-leu-.

The result of amino acid analysis, it was found that fraction To contain the polypeptide fraction To above and proposed in the patent application U.S. N 07/346 181.

A method of OBTAINING a POLYPEPTIDE, CAPABLE of BLOCKING the CALCIUM CHANNELS, by chromatographic separation of the venom of the spider Agelenopsis aperta in the range of 220-230 nm on a column of type VidacRC-18 with a pore size by eluting a mixture of acetonitrile and 0.1% aqueous triperoxonane acid in a gradient increase in the content of acetonitrile in triperoxonane acid, characterized in that use column 22 250 mm, particle size 10 μm, eluting the mixture was fed at a flow rate of 15 ml/min PR is those then collect the fraction 41.5 min elution expose its chromatography on a column of the same type 10 250 mm, particle size 5 μm, elute at a flow rate of 3.5 ml/min by isocratic solvent system of 70% of 0.1% aqueous triperoxonane acid and 30% acetonitrile in the same range, then collect the fraction, loireau 7.5 min, if necessary, subjecting it lyophilization, suspendirovanie in water, lyophilization and obtain the target product with the following features:

- aminobenzene amino acid sequence contains H2N - Lys - Lys - Lys - Cys Jle - Ala - Lys - Asp - Tyr - Gly - Arg - Cys - Lys - Trp - Gly - Gly - Thz - Pro - Cys - Cys - Arg - Gly - Arg - Gly - Cys - Jle - Cys - Ser - Jle - Met - Gly - Tnr - Asn - Cys - Glu - Cys - Lys - Pro - Arg - Leu - Jle - Met - Glu - Gly - Leu-;

molecular weight 5201;

data of mass spectrometry: 0.1 ml of 5% acetic acid (M + 5H)5+ 1041, 20.

 

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