Salt of the transition metal with the polypeptide, and a method of increasing the activity of the polypeptide against hiv

 

(57) Abstract:

The invention relates to compounds salts of the polypeptide represented by the formula (I), where a1AND2AND3AND4X, Y and Z have the values given in the description, and the transition metal, which have high antiviral activity against human immunodeficiency virus. There is also described a method of improving anti-HIV activity of the compounds of the polypeptide of formula (I) consists in the fact that the polypeptide is transferred to the salt with a transition metal, and pharmaceutical composition comprising the compound salt polypetide (I) with a transition metal having antiviral activity. 3 c. and 4 C.p. f-crystals, 2 PL.

The invention relates to a salt of the transition metal with a polypeptide that exhibits a strong affinity for lipopolysaccharides, in particular to endotoxins and in addition, the invention concerns a method of increasing antiviral activity (e.g., anti-HIV activity) of the polypeptide, whereby the specified anti-virus activity is consistently and strongly, by converting the specified polypeptide in the salt of the transition metal, and also relates to pharmaceutical compositions of the ingredient specified salt of the transition metal with the polypeptide (which is sometimes called the salt of the polypeptide and the transition metal).

As shown in the publications referenced below, from horseshoe crabs have identified two families of antimicrobial polypeptides that exhibit affinity to endotoxin.

Cm. for example, Shigenaga et al., 1990, J. Biol. Chem., 265:21350-21354; Kawano et al., 1990, J. Biol. Chem., 265:15365-15367; Muta et al., 1990, J. Biochem., 108:261-266; lined patent application Japan 167230/1990; lined patent application Japan 1152987/1990; lined patent application Japan 53799/1990; U.S. patent number 5068314 (published application with the results of the patent search 500194/1990); Miyata et al., 1989, J. Biochem., 106: 663-668; Akaji et al., 1989; Chem. Pharm. Bull. 37:2661:2664; Tokunaga and Iwanaga, 1989, Taisha (Metabolism), 26:429-439); Shieh et al., 1989, FEBS Lett., 252:121-124; Nakamura et al., 1988, J. Biol. Chem., 263:16709-16713.

One family - the family of theplatinum - allocated from the Japanese sword-bearer Tachypleus. Identified three theplain I, II and III. Another family - a family of polyphemoides - allocated from the American sword-bearer Limulus polyphemus. Identified two polyfusia I and II.

It was found that both these families theplain and Polyphemus inhibit the growth of both gram-positive and gram-negative bacteria at low concentrations, as well as fungi, such as Candida albicans, and form complexes with bacterial lipopolysaccharide (Shigenaga et al., 1990, J. Biol. Chem., 265:wet inhibitory effect on viruses, such as influenza virus, vesicular stomatitis virus (Murakami et al., 1991, Chemotherapy, 37, 327-334) or human immunodeficiency virus (HIV) (Morimoto et al., 1991, Chemotherapy, 37, 206-211).

On the other hand, from the point of view of survival of advanced human beings is extremely necessary to develop drugs that may have a preventive or therapeutic effect against the syndrome of acquired immunodeficiency syndrome (AIDS), caused by infection with human immunodeficiency virus (HIV).

The authors of the present invention as a result of studies on the correlation between the structural transformation of the polypeptide having affinity to endotoxin, and its activity against HIV discovered a series of previously unknown proteins, which differ fundamentally from the conventional structure of the polypeptide swordtails and exhibit a high degree of activity against human immunodeficiency virus (HIV), and these results were published in the following publications (Nakashima et al. , 1992, Antimicrob. Agents Chemother., 36: 1249-1255; Masuda et al., 1992, Biochem. Biophys. Res. Commun., 189:845-850; Tamamura et al. , 1993, Chem. Pharm. Bull., 41:978-980; Tamamura et al., 1993. Biochem. Biophys. Acta, 1163:209-216; Masuda et al., 1992, J. Pharmacobio. Dyn. , 15: s-90; U.S. patent 5571892 (international publication WO 92/04374); U.S. patent 5449752 (what's with expressionism activity against HIV polypeptide, based on the basic structure of the polypeptide obtained from swordtails, which consists of 16 to 18 amino acid residues, the authors present invention was developed and presented as an invention previously unknown concept, which focuses attention on the minimum required structure (international application WO 95/10534).

In accordance with the above invention, the structural concept is taken as an example of compound - polypeptide derived from the polypeptide swordtails as standard material and exhibiting antiviral activity, can be expressed using the following formula [I]

< / BR>
(where A1independently represents the residue of a basic amino acid selected from Lys (lysine), AGD (arginine) and UCP (ornithine); the remainder of the peptide with at least two of these residues essential amino acids; or a residue of N-substituted amino acid or a residue of N-substituted peptide, in which the hydrogen atom in the N-position of the amino acid residue located at the amino-end of the stated principal balance of amino acids or the specified residue of a peptide can be replaced by an acyl group or a group of substituted thiocarbamoyl;

A2independently researched;

AND3independently represents the residue of a basic amino acid selected from Lys, Arg and Orn;

AND4is a HE (originating from the carboxyl group) or-NH2(originating from the group of acid amide);

X represents a peptide residue of the two amino acid residues, where the following position of one of amino acid residue selected from A1A (alanine), Val (valine), Leu (leucine), Il (isoleucine), Sr (serine), Met (methionine) and s (cysteine), one of the amino acids of the A2attached via a peptide bond;

Y represents a peptide residue of the two amino acid residues, which consists of a combination of residue Gly (glycine) and one amino acid residue selected from AND3or a peptide residue two amino acid residues, which consists of a combination of balance Pro (Proline) and one amino acid residue selected from D-Arg, D-Lys and-UCP;

Z is a peptide residue of the two amino acid residues, where the following position of one of amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met, and A2, Cys attached via a peptide bond;

and the rest of the X-Y-Z, connected by peptide bonds attached to each amino acid residue in the 6th and the nternet to each amino acid residue in the 6-m and 10-m regulations through a peptide bond, where a hydrogen atom in the side chain-amino groups of D-Lys, L-Lys, D-Orn or L-Orn, which is the constituent amino acids of Y may be substituted-aminoaniline group.

It has been established that the polypeptide has a structure of the above-mentioned formula [I] , in addition to the high anti-HIV activity can also be given the ability to preserve this activity by modification of specific customers without reducing activity and due to this modification the polypeptide has new properties that allow you to have a wide variety of physical and/or chemical properties and therapeutic use, based on the properties, which has the basic structure, for example, you can increase or decrease the hydrophilicity or lipophilicity, selectively accumulate in a specific tissue, organ or cell, to increase or decrease the retention time in the body or to develop dosage forms with standard dosages. Table 1 shows examples of the polypeptides of the formula [I], which exhibit high activity against HIV.

The authors of the present invention made the invention based on the obtained fact that the formation of a salt between polyphenol polypeptide against HIV and on the basis of the explanation of why the polypeptide shown in the formula [I], specifically active against HIV.

The first aim of the present invention is the creation of a previously non-existing salt of the transition metal with a polypeptide that exhibits high antiviral activity and has a specific structural formula, and the second purpose of the present invention is that by converting the specified polypeptide in the salt of the transition metal to create a way to increase physiological activity, particularly antiviral activity, such as activity against HIV, as well as a way of manifesting pharmaceutically stable activity as a therapeutic agent, as well as to create a medicinal composition.

The present invention relates to compounds of the salt of the transition metal with the polypeptide shown in the formula [I], which exhibits a strong affinity for lipopolysaccharides, in particular to endotoxins and in addition, the present invention relates to a method of increasing antiviral activity (e.g., anti-HIV activity) of the polypeptide, resulting in it is stable and strong, through the transformation specified is soedineniya, which is the salt of the transition metal with the polypeptide shown in the following formula:

< / BR>
(where A1independently represents the residue of a basic amino acid selected from Lys, Arg and Orn; the residue of a peptide having at least two of these residues essential amino acids; or a residue of N-substituted amino acid or a residue of N-substituted peptide, in which the hydrogen atom in the N-position of amino acid residue in the amino-end of the stated principal balance of amino acids or the specified residue of the peptide may be substituted acyl group or a group of substituted thiocarbamoyl;

A2independently represents an amino acid residue selected from Phe, Trp and Tight;

AND3independently represents the residue of a basic amino acid selected from Arg, Lys and Orn;

AND4is a HE (originating from the carboxyl group) or-NH2(originating from the group of acid amide);

X represents a peptide residue two amino acid residues, where the following position of one of amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and Cys, one of the amino acids of the A2attached via a peptide bond;

Y represents a peptide residue of the active ingredient is from a3or a peptide residue two amino acid residues, which consists of a combination of balance Pro and one amino acid residue selected from D-Arg, D-Lys, D-Orn;

Z is a peptide residue two amino acid residues, where the following position of one of amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and AND2, Cys attached via a peptide bond;

and the rest of the X-Y-Z, connected by peptide bonds attached to each amino acid residue in the 6-m and 10-m regulations peptide bonds, or as a result of competitive deletion of X and Z, the rest of Y can be attached directly to each amino acid residue in the 6th and 10th positions of the peptide bonds, where the hydrogen atom in the side chain-amino groups of D-Lys, L-Lys, D-Orn or L-Orn, which is the constituent amino acids of Y may be substituted-aminoaniline group, or acid salts attach the specified connection salts of the transition metal with the polypeptide and acid;

(2) compound salt of the transition metal with the polypeptide or acid salt accession to the specified salt of the transition metal with the polypeptide under item(1), in which the transition metal salt is a complex salt;

(3) compounds salts of the transition IU(1) or (2), in which the transition metal is chosen from the group consisting of iron group comprising Fe, Co and Ni, the group of copper in the composition C, Ad, AI, group of zinc in the composition of the Zn, Cd and LP, the group of manganese in the composition of the MP, TC and Re;

(4) the way to improve and ekspressirovali high and stable anti-HIV activity of the compounds of the polypeptide shown in the following formula:

< / BR>
(where A1independently represents the residue of a basic amino acid selected from Lys, Arg and Orn; the residue of a peptide having at least two of these residues essential amino acids; or a residue of N-substituted amino acid or a residue of N-substituted peptide, in which the hydrogen atom in the N-position of amino acid residue located at the amino-end of the stated principal balance of amino acids or the specified residue of a peptide can be replaced by an acyl group or a group of substituted thiocarbamoyl;

AND2independently represents an amino acid residue selected from Phe, Trp and Tight;

AND3independently represents the residue of a basic amino acid selected from Arg, Lys and UCP;

AND4is a HE (originating from the carboxyl group) or-NH2(originating from the group of acid amide);

X predestinate, selected from Ala, Val, Leu, Ile, Ser, Met and s, one of the amino acids of the A2attached via a peptide bond;

Y represents a peptide residue two amino acid residues, which consists of a combination of residue Gly and one amino acid residue selected from AND3or a peptide residue two amino acid residues, which consists of a combination of balance Pro and one amino acid residue selected from D-Arg, D-Lys, D-Orn;

Z is a peptide residue two amino acid residues, where the following position of one of amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met, and A2, Cys attached via a peptide bond;

and the rest of the X-Y-Z attached by peptide bonds attached to each amino acid residue in the 6-m and 10-m regulations peptide bonds, or by simultaneously operating the deletion of X and Z, the rest of Y can be attached directly to each amino acid residue in the 6th and 10th positions of the peptide bonds, where the hydrogen atom in the side chain-amino groups of D-Lys, L-Lys, D-Orn or L-Orn, which is the constituent amino acids of Y, may be replaced by-aminoaniline group by converting the specified polypeptide [I] in g with transition metal;

(5) the deposits of salt of the transition metal with the polypeptide or an acid salt of joining the specified connection salts of the transition metal with the polypeptide according to (1) and a pharmaceutical carrier;

(6) the composition of p. (5), inhibiting viral activity; and

(7) the composition of p.(5), inhibiting the activity of HIV in a patient.

As examples of acyl groups which may be substituted with a hydrogen atom in the above position N, the above acyl group having from 2 to 18 carbon atoms, and in particular acetyl group, propylaniline group, Butyrina group, hexanoyl group, octonaria group, dekheila group, Laurila group, Mirandolina group, palmifolia group, caarolina group, nicotinoyl group and the like. As examples of substituting groups, which can be used instead of the group of substituted thiocarbamoyl, which, in turn, can replace a hydrogen atom in the above position N is given fluoresceine group, phenyl group, the group of substituted phenyl (for example, dimethylaminophenyl-azafenidin group) and the like.

As examples aminoaniline groups, which may substitute the hydrogen atom in the side chain-aminoaniline group D-Lys, L-Lys, D-Orn or L-Orn, which is a part of the above-mentioned amino acids Y, aminoaniline group having from 2 to 6 atoms in them.

In the present sequence of the polypeptide each symbol represents the balance of amino acid or residue substituted amino acids marked in accordance with internationally accepted three-letter abbreviation, and except where otherwise indicated, the amino acid residue or a residue of a substituted amino acid is an L-shape. For example, each symbol is represented by the following amino acid or substituted amino acid:

Ala, Arg, Cys, Ile, Gly, Leu, Ser, Met, Lys, Orn, Phe, Pro, Trp, Tyr, Val, DArg, DLys, Dorn, Ac-Arg (N--acetylamine), FTC-Arg (N-fluorescein thiocarbamoyl arginine), Laur-Arg (N-- lauroyl arginine), Myr-Arg (N--myristoyl arginine), Nicot-Arg (N--nicotinoyl arginine), Oct-Arg (N--octanol arginine), Parm-Arg (N-Palmitoyl arginine), Parm-Orn (N-Palmitoyl ornithine), PTC-Arg (N--phenylthiocarbamoyl arginine), N-Ac-Dlys (N-aminoacetyl-D-lysine)-N-But-Dlys-D-lysine).

The present invention, which was made on the basis of the above points of view, refers to salts of the polypeptide comprising the salt of a transition metal selected from the group consisting of iron group, copper, group of zinc, and the group of manganese, and the polypeptide shown in the following formula:

< / BR>
(where A1independently represents having, at least two of these residues essential amino acids; or a residue of N-substituted amino acid or a residue of N-substituted peptide, in which the hydrogen atom in the N-position of amino acid residue located at the amino-end of the stated principal balance of amino acids or the specified residue of a peptide can be replaced by an acyl group or a group of substituted thiocarbamoyl;

A2independently represents an amino acid residue selected from Phe, Trp and Tight;

AND3independently represents the residue of a basic amino acid selected from the AGD, Lys and UCP;

AND4is a HE (originating from the carboxyl group) or-NH2(originating from the group of acid amide);

X represents a peptide residue of the two amino acid residues, where the following position of one of amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and s, one of the amino acids of the A2attached via a peptide bond;

Y represents a peptide residue two amino acid residues, which consists of a combination of residue Gly and one amino acid residue selected from AND3or a peptide residue two amino acid residues, which consists of a combination Usc two amino acid residues, where in the following the position of one of amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and AND2, s attached via a peptide bond;

and the rest of the X-Y-Z, attached via a peptide bond, attached to each amino acid residue in the 6-m and 10-m regulations through a peptide bond, or as a result of competitive deletion of X and Z, the rest of Y can be attached directly to each amino acid residue in the 6-m and 10-m regulations through a peptide bond, where the hydrogen atom in the side chain-amino groups of D-Lys, L-Lys, D-Orn or L-Orn, which is the constituent amino acids of Y, may be replaced by-aminoaniline group), and also relates to salts of the specified polypeptide, in which the transition metal salt is a complex salt, and the purpose of the present invention is by converting the specified polypeptide of formula [I] having a high activity against HIV in the product as a salt, to create a way to improve and stabilize the activity of the polypeptide.

Not previously existed compound salt of the transition metal with the polypeptide according to the invention is described below in more detail.

OBTAINING POLYPEPTIDES

The polypeptide shown in the formula [I] of the present from the, ESA, described in "Solid Phase Peptide Synthesis", Stuart&Young, Pierce Chemical Co. , Rockford, Illinois (1984). In the case of N-acylaminoalkyl or N--elliptica, where the hydrogen atom in the N-position to the amino end of residue amino acid substituted acyl group in A1formula [I] , a polypeptide with a straight chain of the formula [I] is associated with an insoluble polymer, to obtain a polypeptide polymer, and carry out the reaction of the specified polypeptide polymer with carboxylic acid anhydride or carboxylic acid corresponding to the acyl group, using a condensing agent, in order to allievate specified N-terminal amino group and get N-acylated polypeptide polymer. Then this insoluble polymer and a protective group of amino acids is removed and receive a polypeptide with a straight chain of the formula [I]. In the case of N--substituted thiocarbamoyl amino acid or a residue of N-substituted thiocarbamoyl peptide, where the hydrogen atom in the N-position to the amine end of the amino acid residue is replaced thiocarbamoyl group in A1formula [I], N-terminal N-substituted thiocarbamoyl polypeptide according to the invention can be obtained by reaction of the above-mentioned polypeptide with the compound by Braz polypeptide carboxyl end of the amino acid residue in the 14th position can be either free (A4matches-HE), or converted into acid amide (A4corresponds to the-NH2).

Except where otherwise noted, individual amino acids used in the above method, solid-phase synthesis, are L-shaped, and the essential amino acid, coupled with Proline in the 8th position, denoted by Y, is limited to the D-form.

Alternatively, the polypeptide according to the invention can be also obtained by a method comprising the use of recombinant DNA. In accordance with this method, sequence, nucleotides encoding for a polypeptide according to the invention, it is possible to clone and Express through methods well-known to specialists in this field of technology.

Cm. for example, Maniatis et al., Molecular Cloning, A Laboratory anual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1991.

The polypeptide according to the invention can be extracted and purified by using techniques known in the field of technology-related polypeptides, such as extrace, recrystallization, various types of chromatography (gelfiltration, ion exchange, distribution, adsorption chromatography, chromatography with reversed phase), electrophoretogram high-resolution reversed-phase.

As examples of works that describe such manufacturing methods, you can refer to the following publications: U.S. patent 5571892 (international application WO 92/04374); U.S. Patent 5449752 (published patent application Japan 163298/1993) and international publication WO 95/10534.

The polypeptide shown in the formula [I], contains in its molecule a residue cysteine, arginine and lysine, and according stereostructures information, at least one pair of cysteine residues or residues of arginine adopt such a conformation that the side chain-SH or guanidine side chain is located directly on the same side in stereostructure, so that the mentioned side chain capable of forming a complex salt with the compound of the transition metal. However, this polypeptide usually takes oxidized form-S-S, so it is preferable that the specified polypeptide changed to pre-reduced (-SH) form, which will facilitate the formation of a complex salt with a transition metal.

For example, a complex salt with a transition metal can be obtained by adding to an aqueous neutral solution of specified reduced forms of the polypeptide, at least two equivalents calichescot acid (for example, zinc acetate).

Compound salt of the transition metal with the polypeptide shown in the formula [I] of the present invention, showing an extremely strong basicity.

Thanks to this strong basicity salt accession form by adding acid. Form a salt with a pharmaceutically suitable acid, for example with inorganic acid, such as hydrochloric acid, Hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid and the like, with an organic carboxylic acid, such as acetic acid, with halogenosilanes acetic acid, such as triperoxonane acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid and the like, acidic sugars such as glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid and the like, acidic polysaccharides, sometimes involving sulfate polysaccharides, such as hyaluronic acid, chondroitin sulfates, alginic acid and the like, with organic sulfonic acids such as methanesulfonate acid, p-toluensulfonate acid and the like. Salt prigotovleniya medicinal compositions as salts of accession with the specified pharmaceutically suitable acid.

THE USE OF COMPOUNDS OF THE SALT OF THE TRANSITION METAL WITH THE POLYPEPTIDE ACCORDING TO THE INVENTION

Compound salt of the transition metal with the polypeptide of formula [I] has the ability to bind endotoxins, has antibacterial activity and ability to demolitioning the formed elements of blood are sensitive to endotoxins. In addition, the compound salt of the transition metal with the polypeptide according to the invention has very high antiviral activity. In a specific embodiment of the invention the compound of salt of the transition metal with the polypeptide according to the invention have activity against HIV. Medicine, in particular the tool against HIV, according to the invention can be prepared in the form of a pharmaceutical composition comprising the compound of salt of the transition metal with the polypeptide shown in the formula [I] or the salt of the accession of the compounds of the salt of the transition metal and the polypeptide with a pharmaceutically suitable acid as an effective component, as well as pharmaceutically suitable carrier selected in accordance with the method of appointment and method of reception l is overestimate buffers, such as solutions Hanks or ringer's, saline, isotonic glucose solution or a mixture thereof, and heparinised solution of citric acid, sodium citrate and dextrose. The remedy for HIV according to the invention is prescribed for oral administration or for parenteral administration, depending on the object against which treatment, or for disinfection against viral diseases inside the body, or for disinfection of infected external parts of the body such as the surface of the body, and it can be prepared in such a preparative form as powder, granules, solution for injection or for oral administration, tablets, suppositories, pessaries, ointment, cream or spray, using a suitable pharmaceutically suitable carrier, in accordance with the method of appointment and taking medicines.

When the tool against HIV according to the invention administered to a patient directly in the form of injections, the polypeptide or its salt according to the invention are dissolved in saline and injected continuously or intermittently in an amount of from 10 to 5000 mg per kg of body weight per day or by intravenous infusion through a drip.

EXAMPLES

Below p is ASS="ptx2">

In the following examples describe examples of the preparation of salts of the transition metal with the polypeptide (1) and polypeptide (2) and the results of tests for activity against HIV salts of the transition metal with the polypeptide according to the invention, as well as to known polypeptides with affinity to endotoxin.

EXAMPLE 1: OBTAINING COMPLEX SALT OF ZINC WITH THE POLYPEPTIDE (1)

The following polypeptide (1) (see the end of the description) was synthesized and obtained using the method described in U.S. patent 5571892 (international publication WO 92/04374) and in U.S. patent 5449752 (published patent application Japan 163298/1993).

1.1. OBTAINING A RESTORED FORM OF THE POLYPEPTIDE (1)

Acetate polypeptide (1) (10,2 mg, 3,37 mmol), obtained in accordance with the above-mentioned international publication PCT was dissolved in purified water (0.5 ml). To this solution was added dithiothreitol (production Corporation Seikagaku) (26,0 mg, 169 μmol), 50-fold equivalent relative to the polypeptide (1), washed with gaseous nitrogen and stirred in a stream of nitrogen at room temperature for two hours. Move the specified response recovery was monitored by HPLC to confirm the completion loaded into a column (2.5 x 70 cm) with Sephadex G-25 (fine), (production company Pharmacia biotech. TOoLtd.), pre-balanced aqueous solution of 25% acetic acid, extracted from the adsorbent using the same aqueous solution of 25% acetic acid and then subjected to fractionation (1 faction = 224 drops). The fractional portion of number fractions 26 and 27, which showed a positive reaction to Ellman (G. L. Ellman, Arch. Biochem. Biophys. , 82, 70 (1959); the method of detection of thiol groups) and the reaction fluorescence (A. M. Felix et al., J. Chromatogr., 89, 361 (1974), the method of fluorescent detection of amino groups), collected a solution of the indicated fractions were concentrated under vacuum and, after dilution with an aqueous solution of 10% acetic acid, the solution was subjected to lyophilization and as a result received the desired acetate restored form of the polypeptide (1), as required.

Output: 6,8 mg (67%).

1.2. ANALYSIS OF REDUCED FORMS OF THE POLYPEPTIDE (1)

Acetate restored form of the polypeptide (1) obtained in section 1.1, were subjected to acid hydrolysis in 4 M methanesulfonic acid containing 0.2% tryptamine, at 115oWith in 24 hours, according to the method of Liu and others (T.-Y. Liu et al. , J. Biol. Chem., 251, 1936 (1976)). Its amino acid composition agreed well with am is SUP>D obtained acetate restored form of the polypeptide (1) was -26,0o(C = 0,09, an aqueous solution of 1M acetic acid).

Moreover, the obtained acetate restored form of the polypeptide (1) showed a single peak when analyzed by HPLC under the following conditions.

THE CONDITIONS OF THE ANALYSIS BY HPLC AND THE RESULTS

Column: TSK-gel ODS - 120 T (0,46 15 cm) (Toso TO0., Ltd) + TSK hard gel ODS - 120 T (0,32 1,5 cm) (Toso TO0., Co., Ltd.).

Gradient elution:

Eluent:

10% acetonitrile / 0.1% of triperoxonane acid (solution A).

80% acetonitrile / 0.1% of triperoxonane acid (solution B).

The terms of the gradient:

The time gradient: min 1,0; 29,4 min; 35,0 minutes

The concentration of the solution In respectively: 0%; 42%; 100%.

The concentration of the solution And therefore: 10%; 39,4%; 80%.

Temperature: 40oC.

Flow rate: 0.8 ml/min

Detection: 220 nm and 280 nm.

Used quantity: 5 ál

(concentration of peptide: 1 mg/ml).

The time of elution:

acetate restored form of the polypeptide (I): 19,27 min,

acetate polypeptide (I): 18,24 minutes

1.3. Get complete Solusinya in section 1.1., was dissolved in purified water or 1M buffer solution of ammonium acetate (pH 7.2).

To this aqueous solution or buffer solution was added an aqueous solution of 0.005 M of zinc acetate, corresponding to two equivalents of ion zinc (II) with respect to the restored form of the polypeptide (1). The final concentration of the polypeptide brought to 5 mg/l and received a comprehensive solution reduced forms of the polypeptide (1) and zinc ions (II).

1.4. CONFIRMATION OF THE STRUCTURE OF COMPLEX SALT REDUCED FORMS OF THE POLYPEPTIDE (1) AND ION ZINC (II) USING ION-SPRAY MASS SPECTROMETRY

A portion of an integrated solution reduced forms of the polypeptide (1) and zinc ions (II), obtained in section 1.3, was used as a test solution for the structural analysis and carried out a structural analysis by ion-spray mass spectrometry under the following conditions.

CONDITIONS OF THE ION-SPRAY MASS SPECTROMETRY

Equipment: three-stage quadrupole mass spectrometer of the type AR IIIE (manufacturer - Perkin-Elmer Siex TO0., Ltd., service - Takara Shuso TO0., Co., Ltd.).

Infusion of the sample: for infusion sample with flow rate 5 µl/min COI is agme: 130 volts.

The region of the mass spectra and the like: weight against electric charge (m/z) 600 - 1800

(in positive ion mode, in increments of 0.5 atomic units of mass, an average of 30 cycles of the scan).

Analysis of obtained data was performed using s 3/22 (Sciex).

THE RESULTS OF ION-SPRAY MASS SPECTROMETRY

Measured mass values for state charges +2 and +3 (m/z: 1277,00 and 851,67 respectively), and the reconstructed mass value (m/z: 2552,48) agreed well with the calculated mass value for the recovered polypeptide (1) + Zn - 4N (m/z: 2552,40).

In other words, it was shown that the polypeptide (1) and ion zinc (II) form a complex in which the ratio of peptide: Zn is 1:1.

DISCUSSION

It was reported that the structure of the complex consisting of the peptide or protein and metal, can be identified using ion-spray mass spectrometry or electrospray mass spectrometry. For example, see the analysis of the zinc finger domain structure of the DNA binding of the glucocorticoid receptor (H. E. Witkowska et al., J. Amer. Chem. Soc., 117, 3319 (1995)), analysis of the coordination patterns of copper relative to zinc finger region of the protein (T. W. Hutchens et al., FEBS Lett., 309, 170 (1992)), review Umeda and what the experiment was also established, what is the structure of the complex of restored form of the polypeptide (1) and zinc ions (II) can be identified using ion-spray mass spectrometry.

1.5 CONFIRMATION OF THE STRUCTURE OF COMPLEX SALT REDUCED FORMS OF THE POLYPEPTIDE (1) AND ION ZINC (II) WITH ULTRAVIOLET ADSORPTION SPECTROPHOTOMETRY

Portions of an aqueous solution of the restored form of the polypeptide (1) obtained in R. 1.1, and an integrated solution reduced forms of the polypeptide (1) and zinc ions (II), obtained in section 1.3, was used as test solution for structural analysis, and structural analysis was performed by measuring the ultraviolet absorption spectra and their differential spectra in the following conditions.

MEASUREMENT OF THE ULTRAVIOLET ABSORPTION SPECTRA

Equipment: spectrophotometer UV-visible radiation Ubest 30 (Nihon-Bunko TO0., Co., Ltd.).

The measured wavelength: 200 - 340 nm.

Sample: after placing each freshly prepared test sample in the measuring tube was flushed with gaseous nitrogen and a measurement was performed immediately.

Normal spectra ultraviolet absorption: is eptide (1) and the integrated solution reduced forms of the polypeptide (1) and zinc ions (II).

Differential UV spectra:

Control: the solution of the reduced forms of the polypeptide (1).

Test sample: an integrated solution reduced forms of the polypeptide (1) and zinc ions (II).

THE RESULTS OF MEASUREMENTS OF THE SPECTRA OF THE ULTRAVIOLET ABSORPTION

Normal spectra ultraviolet absorption:

The solution of the reduced forms of the polypeptide (1):

Demonstrated the presence of a large peak absorption at 250-300 nm, which is typical for the absorption caused by tryptophan - an amino acid that is incorporated into the polypeptide (1). Showed a maximum absorption at 278 nm and the shoulder of the spectral line at 280 nm, which are characteristic absorption due to tryptophan.

Integrated solution reduced forms of the polypeptide (1) and zinc ions (II):

Received the absorption curve, which was broadened in relation to the curve of the absorption of the solution above the restored form of the polypeptide (I), with a range from 250 to 200 nm. In order to clarify the wavelengths of absorption, made measurements the following differential spectrum.

Differential UV range:

Got th differential ABSORPTION

As shown above, adding zinc ions (II) to the restored form of the polypeptide (1) ultraviolet absorption is widened mainly in the range from 215 to 235 nm.

This suggests, as discussed below, that in solution the restored form of the polypeptide (1) and zinc ions (II) SH-group of cysteine reduced forms of the polypeptide (1) forms a complex with the zinc ion through mercaptide link.

DISCUSSION

It is known that metallothionein, a protein related to detoxification of heavy metal, has a structure in which the SH group which is part of the cysteine residue forms a complex with the ion cadmium (II), zinc (II), copper (I, II), mercury (II) or similar through mercaptide communication, and when the metal binds to APO-metallothionein (SH-form), ultraviolet absorption increases in the range of the spectrum with wavelengths characteristic of mercaptides connection of each metal. For example, it is known that in the case mercaptide complex communication with the ion zinc (II) ultraviolet absorption increases and is widened mainly in the range of 220-230 nm (see J. H. R. Kagi and B. L. Vallee, J. Biol. Chem., 236, 2435 (1961); M. Vasak et al., Biochemistry, 20, 2852 (1981); A. R. Thrower et al., J. Biol. Chem., 263, 7037 (1988); J. H. R. Kagi et al., Environmen is s (220-230 nm) due mercaptides communication of this ion zinc (II) is consistent with increased wavelength range differential range, observed for the solution above the restored form of the polypeptide (1) and zinc ions (II).

In other words, found that in the solution of the reduced forms of the polypeptide (1) and zinc ions (II) S-group of cysteine reduced forms of the polypeptide (1) forms mercaptide communication with the ion zinc (II).

1.6 ACTIVITY AGAINST HIV COMPLEX SALTS OF REDUCED FORMS OF THE POLYPEPTIDE (1) AND ION ZINC (II)

A portion of an integrated solution reduced forms of the polypeptide (1) and zinc ions (II) obtained in paragraph 1.3, was used as test solution for measuring anti-HIV activity and measured the activity against HIV. EXAMPLE 2: OBTAINING COMPLEX SALT OF ZINC WITH POLYPEPTIDE (2)

Polypeptide (2) (see the end of the description), shown in the following formula were obtained according to the method described in international PCT publication WO 95/10534.

2.1 OBTAINING THE RESTORED FORM OF THE POLYPEPTIDE (2)

Acetate polypeptide (2) (10.0 mg, of 3.94 mmol), obtained in accordance with the above-mentioned international publication PCT was dissolved in purified water (0.5 ml). To this solution was added dithiothreitol (32,0 mg, 207,5 µmol) (production Corporation Seikagaku) 53-fold equivalent is th temperature for two hours.

The specified flow of reduction reaction was monitored by HPLC and confirmed the full recovery.

After completion of the reduction reaction of the specified reaction solution was loaded onto a column (2.5 x 70 cm) with Sephadex G-25 (fine), (production company Pharmacia biotech. TO0., Ltd.), pre-balanced aqueous solution of 25% acetic acid, extracted from the adsorbent using the same aqueous solution of 25% acetic acid and then subjected to fractionation (1 faction = 224 drops). The fractional portion of number fractions 26 and 27, which showed a positive reaction in the analysis using the reaction of Ellman and using reaction fluorescence was collected, the solution of the indicated fractions were concentrated in vacuum, and after dilution with an aqueous solution of 10% acetic acid, the solution was subjected to lyophilization, resulting in received acetate restored form of the polypeptide (2), as required.

Output: 9,8 mg (98%).

2.2 ANALYSIS OF REDUCED FORMS OF THE POLYPEPTIDE (2)

Acetate restored form of the polypeptide (2) obtained in section 2.1, were subjected to acid hydrolysis in 4 M methanesulfonic acid containing 0.2% tryptamine 1.2. Its amino acid composition agreed well with the amino acid composition of the reduced forms of the polypeptide (2).

Specific optical rotation []20D obtained acetate restored form of the polypeptide (2) was -23,3o(C = 0,04, an aqueous solution of 1M acetic acid).

Moreover, the obtained acetate restored form of the polypeptide (2) showed a single peak when analyzed by HPLC, carried out under the same conditions as the HPLC analysis, performed for the polypeptide (1) in section 1.2.

THE RESULTS OF HPLC ANALYSIS

The time of elution:

acetate restored form of the polypeptide (2) - 16,14 min

acetate polypeptide (2) $ 15.87 with min

2.3. THE COMPLEX SALT OF THE RESTORED FORM OF THE POLYPEPTIDE (2) AND ION ZINC (II)

Acetate restored form of the polypeptide (2) obtained in section 2.1, was dissolved in purified water or 1M buffer solution of ammonium acetate (pH 7.2).

To this aqueous solution or buffer solution was added an aqueous solution of 0.005 M of zinc acetate, corresponding to one equivalent of zinc ion (II) with respect to the restored form of the polypeptide (2). The final concentration of the polypeptide brought up to 5 mg/l and received comp COMPLEX SALTS of reduced FORMS of the POLYPEPTIDE (2) AND ION ZINC (II) USING ION-SPRAY MASS SPECTROMETRY

A portion of an integrated solution reduced forms of the polypeptide (2) and ion zinc (II), obtained in section 2.3, was used as test solution for structural analysis, and performed structural analysis by ion-spray mass spectrometry, under the same conditions under which the spent ion-spray mass spectrometry integrated solution reduced forms of the polypeptide (1) and zinc ions (II), as described in section 1.4.

THE RESULTS OF ION-SPRAY MASS SPECTROMETRY

Observed mass values for state charges +2 and +3 (m/z: 1030,50 and 687,34 respectively), and the reconstructed mass value (m/z: 2059,98) agreed well with the calculated mass value for the recovered polypeptide (2) +Zn-4H (m/z: 2059,83).

In other words, showed that the recovered polypeptide (2) and ion zinc (II) form a complex in which the ratio of peptide : Zn is 1:1.

2.5 CONFIRMATION OF THE STRUCTURE OF COMPLEX SALT REDUCED FORMS OF THE POLYPEPTIDE (2) AND ION ZINC (II) BY UV ADSORPTION SPECTROPHOTOMETRY

Portions of an aqueous solution of the restored form of the polypeptide (2) obtained in section 2.1, and comprehensive solution Voss is the solution for the structural analysis, and structural analysis was performed by measuring the ultraviolet absorption spectra and their differential spectra in the same conditions that were used for the polypeptide (1) in R. 1.5.

MEASUREMENT OF THE ULTRAVIOLET ABSORPTION SPECTRA

Normal spectra ultraviolet absorption:

Control: distilled water.

Test solution: the solution is reduced forms of the polypeptide (2) and the solution of complex salt reduced forms of the polypeptide (2) and zinc ions (II).

Differential UV spectra:

Control: the solution of the reduced forms of the polypeptide (2).

Test solution: an integrated solution reduced forms of the polypeptide (2) and zinc ions (II).

THE RESULTS OF MEASUREMENTS OF THE SPECTRA OF THE ULTRAVIOLET ABSORPTION

Normal spectra ultraviolet absorption:

The solution of the reduced forms of the polypeptide (2): found the presence of a large peak absorption at 250 - 300 nm, which is typical for the absorption caused by tryptophan - an amino acid that is incorporated into the polypeptide (2). Showed a maximum absorption at 278 nm and the shoulder of the spectral line at 280 nm, which is characteristic the Chida (2) and ion zinc (II): got a curve of absorption, which was broadened in relation to the curve of the absorption of the solution above the restored form of the polypeptide (2), with a range from about 250 to 200 nm.

Differential UV range:

Received differential spectrum with a peak in the range from 215 to 235 nm and the difference absorption.

ANALYSIS OF THE OBTAINED SPECTRUM OF THE ULTRAVIOLET ABSORPTION

As was shown above, adding zinc ions (II) to the restored form of the polypeptide (2) observed that the UV absorption is widened mainly in the range from 215 to 235 nm.

Respectively, showed that in solution the restored form of the polypeptide (2) and ion zinc (II) SH-group of cysteine reduced forms of the polypeptide (2) forms a complex with the zinc ion through mercaptide link.

2.6 ACTIVITY AGAINST HIV COMPLEX SALTS OF REDUCED FORMS OF THE POLYPEPTIDE (2) AND ION ZINC (II)

A portion of an integrated solution reduced forms of the polypeptide (2) and ion zinc (II) obtained as described above was used as test solution for measuring anti-HIV activity and measured the activity against HIV.

EXAMPLE 3: ANTIVIRAL ACTIVITY AGAINST Viruslocker in accordance with example 1, and a complex of zinc with the polypeptide of (2) obtained in accordance with example 2, was tested and was determined according to the following method.

In 96-well microtiter tablet directly after infection has placed HIV-infected cells MT-4 (2.5 x 104cells/well, multiplicity of infection: 0,001), along with participant materials in different koncentracija. After incubation in the incubator with CO2at 37oC for 5 days was determined by the number of surviving cells, using the MTT method (Pauwels et al., J. Virol. Methods 20, 309-321 (1998)). Antiviral activity was expressed as the concentration that prevented 50% cell death caused by HIV infection (EC50: concentration with 50% efficiency). On the other hand, in order to determine the cytotoxicity of the tested substances to cells MT-4, was cultivated not virus-infected cells together with participant materials in different concentrations, as described above. Cytotoxicity was expressed as the concentration that causes 50% cytotoxicity (CC50), due to the examinees materials, and the approximate ratio SS50to EC50meant as an effective ratio (SI).

The results of the activity measurements of Protista

ID sequence number: 1

Sequence length: 14

Sequence type: amino acid

Topology: linear

Molecular type: peptide

Feature:

Location: 1

Other information:

XAA independently represents the residue of a basic amino acid selected from lysine, arginine and ornithine; residue peptide having at least two of these residues essential amino acids; or a residue of N-substituted amino acid or a residue of N-substituted peptide, in which the hydrogen atom in the N-position of the amino acid residue at the amino-end of the stated principal balance of amino acids or the specified residue of the peptide may be substituted acyl group or a group of substituted thiocarbamoyl.

Location: 2

Other information:

XAA independently represents the residue of an amino acid selected from phenylalanine, tryptophan and tyrosine.

Location: 4

Other information:

XAA independently represents the residue of an amino acid selected from phenylalanine, tryptophan and tyrosine.

Location: 5, 6

Other information:

XAA independently represents an OST which I info:

XAA independently represents a peptide residue of the two amino acid residues, in which the following position one amino acid residue selected from alanine, valine, leucine, isoleucine, serine, methionine and cysteine, one of the following amino acids phenylalanine, tryptophan or tyrosine is attached via a peptide bond.

Location: 8

Other information:

XAA is a peptide residue of the two amino acid residues, which consists of a combination of glycine residue and remainder of one amino acid selected from arginine, lysine and ornithine or a peptide residue two amino acid residues, which consists of a combination of Proline residue and remainder of one amino acid selected from D-arginine, D-lysine, D-ornithine.

Location: 9

Other information:

XAA is a peptide residue of the two amino acid residues, in which the following position of one of amino acid residue selected from alanine, valine, leucine, isoleucine, serine, methionine, phenylalanine, tryptophan and tyrosine, cysteine is attached via a peptide bond.

Location: 7, 8, 9

Other information:

The residue XAA-XAA-XAA, Dag peptide bond, or because at the same time acting deletions XAA in 7th position and Haa in the 9th position, the remainder of the Haa in the 8th position can be attached directly to each amino acid residue in the 6-m and 10-m regulations, through a peptide bond, where the hydrogen in the side chain-amino groups of D-lysine, L-lysine, D-ornithine or L-ornithine, which is the constituent amino acids of the 8th XAA can be substituted-aminoaniline group.

Location: 10

Other information:

XAA independently represents the residue of an amino acid selected from phenylalanine, tryptophan and tyrosine.

Location: 11, 12

Other information:

XAA independently represents the residue of a basic amino acid selected from arginine, lysine and ornithine.

Location: 14

Other information:

XAA independently represents the residue of a basic amino acid selected from arginine, lysine and ornithine.

1. Compound salt of the transition metal with the polypeptide shown in the following formula:

< / BR>
(Identification sequence: 1), in which

A1independently represents the residue of a basic amino acid selected from Lys, Arg and Orn, or the remainder peptidase a balance of amino acids, selected from Phe, Trp and Tight;

AND3independently represents the residue of a basic amino acid selected from Lys, Arg and Orn;

AND4is a HE (originating from the carboxyl group) or-NH2(originating from the group of acid amide);

X represents a peptide residue two amino acid residues, where the first amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and Cys, and the second amino acid residue selected from A2attached through a peptide bond;

Y represents a peptide residue of the two amino acid residues, which consists of a combination of a Gly residue and remainder of the amino acids selected from AND3or a peptide residue two amino acid residues, which consists of a combination of balance Pro and one amino acid residue selected from D-Arg, D-Lys, D-Orn;

Z is a peptide residue of the two amino acid residues, where one amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and AND2, Cys attached through a peptide bond; and the remainder of the X-Y-Z, connected by peptide bonds attached to each amino acid residue in the 6-m and 10-m regulations peptide bonds, or as a result of competitive deletion of X and Z, the rest of Y can be prisoedinenie salt of the transition metal with the polypeptide according to p. 1, in which the transition metal salt is a complex salt.

3. Compound salt of the transition metal with the polypeptide under item 1 or 2, in which the transition metal is chosen from the group consisting of iron group comprising Fe, Co and Ni, from the group of copper in the composition C, Ad, AI, from the group of zinc in the composition of the Zn, Cd and LP and from the group of manganese in the composition of the MP, TC and Re.

4. A method of improving anti-HIV activity of the compounds of the polypeptide shown in the following formula:

< / BR>
(Identification sequence: 1), in which

A1independently represents the residue of a basic amino acid selected from Lys, Arg and Orn, or a residue of a peptide having at least two of these residues essential amino acids;

AND2independently represents an amino acid residue selected from Phe, Trp and Tight;

AND3independently represents the residue of a basic amino acid selected from Lys, Arg and Orn;

AND4is a HE (originating from the carboxyl group) or-NH2(originating from the group of acid amide);

X represents a peptide residue two amino acid residues, where the first amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and Cys, and a second amino the economic balance of the two amino acid residues, which consists of a combination of a Gly residue and remainder of the amino acids selected from AND3or a peptide residue two amino acid residues, which consists of a combination of balance Pro and one amino acid residue selected from D-Arg, D-Lys, D-Orn;

Z is a peptide residue of the two amino acid residues, where one amino acid residue selected from Ala, Val, Leu, Ile, Ser, Met and AND2, Cys attached through a peptide bond; and the remainder of the X-Y-Z, connected by peptide bonds attached to each amino acid residue in the 6-m and 10-m regulations peptide bonds, or as a result of competitive deletion of X and Z, the rest of Y can be attached directly to each amino acid residue in the 6-m and 10-m regulations peptide bonds, consisting in that the polypeptide (I) was transferred to the salt with a transition metal.

5. Pharmaceutical composition having antiviral activity comprising an effective amount of the compound of salt of the transition metal with the polypeptide under item 1, and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to p. 5, inhibiting viral activity.

7. The pharmaceutical composition according to p. 5, inhibiting activity VI

 

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