RU2473559C1 - Peptide azemyopsyn selectively reacting with nicotine muscarinic receptors and applicable as myorelaxant in medicine and cosmetology - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to nicotinic acetylcholine receptor blocking biologically active peptides which can find application in biochemistry, biotechnology and medicine. The peptide azemyopsin has an amino acid sequence: H₂N-Asp¹-Asn²-Trp³-Trp⁴-Pro⁵-Lys⁶-Pro⁷-Pro⁸-His⁹-Gln¹⁰-Gly¹¹ Pro¹²-Arg¹³-Pro¹⁴-Pro¹⁵-Arg¹⁶-Pro¹⁷-Arg¹⁸-Pro¹⁹-Lys²⁰-Pro²¹-COX wherein X is a group OH or NH₂, and has an ability to react selectively with nicotine muscarinic receptors. The peptide may be used an a base for preparing myorelaxants selectively blocking muscarinic receptors.

EFFECT: invention presents a new type of natural toxins reacting with nicotine receptors and containing no disulphide bonds.

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Description

The invention relates to biochemistry, specifically to biologically active polypeptides that have the ability to selectively interact with nicotinic muscle cholinergic receptors and can be used in scientific research or medicine.

Nicotinic cholinergic receptors (nhr) comprise a large family of ligand-controlled ion channels. NHRs are involved in the processes of fast synaptic transmission in the neuromuscular joints or between neurons. [Wells GB. Structural answers and persistent questions about how nicotinic receptors work. // Front Biosci - 2008. - vol.13 - p.5479-5510]. In addition, it is known that neural HCR is also present in non-neuronal cells, where they play an essential functional role [Grando SA. Basic and clinical aspects of non-neuronal acetylcholine: biological and clinical significance of non-canonical ligands of epithelial nicotinic acetylcholine receptors. // J Pharmacol Sci. - 2008 .-- vol. 106. - p.174-179]. For example, in the immune system, non-neural NHRs play an important role in regulating inflammation. There are two large groups of NHR: muscle type receptors and neural receptors [Dani JA, Bertrand D. Nicotinic acetylcholine receptors and nicotinic cholinergic mechanisms of the central nervous system. // Annu Rev Pharmacol Toxicol. - 2007 - vol. 47 - p.699-729]. muscle-type NCHRs consist of five subunits (two alpha1, one beta1, one gamma (or epsilon) and one delta), which are pseudo-symmetrically located around a central axis that coincides with the ion channel of the receptor. Two binding sites of agonists or competitive antagonists are located at the contact sites of two alpha subunits with neighboring gamma and delta subunits. Neural NHRs are composed of two different types of subunits: alpha (alpha2-alpha10) and beta (beta2-beta4). These receptors exist in the form of hetero-oligomeric (formed by various combinations of alpha and beta subunits) or homo-oligomeric (formed by alpha-subunits of alpha7-alpha10 subtypes) proteins. All the above subtypes of nhr differ not only structurally, but also pharmacologically. A detailed study of the functioning of the various subtypes of NHR requires the use of accurate and selective biochemical instruments. Such tools are, in particular, neurotoxins of snake venoms, which can selectively interact with some subtypes of nhr. The so-called short-type alpha-neurotoxins (59-63 amino acid residues, four disulfide bonds) block only muscle-type nhr, while long-type neurotoxins (66-74 amino acid residues, five disulfide bonds) also block the neuronal homopentameric alpha7-alpha9 subtypes. [Tsetlin V, Utkin Y, Kasheverov I. Polypeptide and peptide toxins, magnifying lenses for binding sites in nicotinic acetylcholine receptors. // Biochem Pharmacol. - 2009 - vol. 78 - p. 720-731]. In long-type neurotoxins, a fifth disulfide bond is absolutely necessary for interaction with neural cholinergic receptors. Neither these nor short type neurotoxins interact with neuronal heteropentameric nchr. There is another group of toxins, the so-called kappa-bungarotoxins, which selectively and efficiently block the neural NHR of the alpha3beta2 subtype and to a lesser extent the alpha4beta2 and alpha7 subtype. Structurally, kappa-bungarotoxins are similar to long-type alpha-neurotoxins, but exist as non-covalent dimers [Dewan JC, Grant GA, Sacchettim JC. Crystal structure of kappa-bungarotoxin at 2.3-A resolution. // Biochemistry. - 1994 .-- vol. 33. - p.13147-13154].

In the early eighties of the last century, peptide toxins were discovered in the mollusks of the genus Conus, causing a postsynaptic block and called alpha-conotoxins [Kasheverov IE, Utkin YN, Tsetlin VI. Naturally occurring and synthetic peptides acting on nicotinic acetylcholine receptors. // Curr Pharm Des. 2009 .-- vol.15. - p.2430-2452]. To date, about 50 of these toxins have been isolated and characterized. Functionally, all of them are competitive antagonists of NHR, however, they differ in the selectivity of interaction with certain subtypes of receptors. Two main groups of alpha-conotoxins can be distinguished, one includes toxins interacting with muscle NHR - muscle conotoxins, and the other - interacting with neural NHR - neuron conotoxins. The latter usually interact with two or more subtypes of NHR. A characteristic feature of muscle conotoxins is their ability to distinguish between two binding sites on Torpedo or muscle type NHP. By structure, alpha-conotoxins are peptides 12-20 amino acid residues long and contain 2 disulfide bonds. Due to the small size of conotoxins and their analogues can be obtained by peptide synthesis, however, the presence of several disulfide bonds significantly reduces the yield of the target product due to the formation of by-products with improper closure of disulfide bonds. These peptides have prospects for use in pharmacology. So, conotoxin Vc1.1 (also known as ACV1) is being tested as a means of combating pain. This toxin acts on neural NHR. Its effect was studied in vivo on several animal models reproducing human neuropathic pains [Livett BG, Sandall DW, Keays D., Down J., Gayler KR, Satkunanathan N., Khalil Z. Therapeutic applications of conotoxins that target the neuronal nicotinic acetylcholine receptor . // Toxicon. - 2006 .-- vol. 48. - p.810-829].

Closest to the claimed peptide by properties are peptides of vaglerin from the poison of South Asian kufiya Tropidolaemus wagleri. They consist of 22-24 amino acid residues and contain one disulfide bond [Weinstein SA, Schmidt JJ, Bernheimer AW, Smith LA. Characterization and amino acid sequences of two lethal peptides isolated from venom of Wagler's pit viper, Trimeresurus wagleri. // Toxicon. - 1991 .-- vol. 29. - p.227-236]. These toxins interact with muscle-type nhr, while they effectively interact with mature receptors containing the epsilon subunit, but not with embryonic nhr containing the gamma subunit [McArdle JJ, Lentz TL, Witzemann V, Schwarz H, Weinstein SA, Schmidt JJ. Waglerin-1 selectively blocks the epsilon form of the muscle nicotinic acetylcholine receptor. // J Pharmacol Exp Ther. - 1999 .-- vol. 289. - p. 543-550].

The peptides described above, interacting with muscle-type HXR, block muscle contraction. This property can be used to prepare muscle relaxants [Olivera BM, Layer RT, Watkins M, Hillyard DR, McIntosh JM, Schoenfeld R, Jones RM, Nielsen J. Alpha-conotoxin peptides. WO / 2002/007750]. Recently, data have appeared on the possibility of using peptides interacting with muscle NHR for cosmetic purposes. The inclusion of such compounds in cosmetic preparations (creams) leads to smoothing of wrinkles [Garcia Sanz N, van den Nest W, Carreno Serraima C, Ferrer Montiel A, Cebrian Puche J, Alminana Domenech N, Fernandez Ballester G. Compounds which inhibit muscle contraction. WO / 2011/009626].

All neurotoxins described above contain disulfide bonds. Snake neurotoxins containing more than 60 amino acid residues and 4-5 disulfide bonds are quite complex for chemical synthesis. Konotoxins and vaglerins can be synthesized by peptide synthesis, however, the presence of disulfide bonds and the need for their correct closure significantly reduces the yield of the target product.

The invention solves the problem of expanding the range of peptides selectively interacting with muscle type nicotinic cholinergic receptors.

The problem is solved by the peptide azemiopsin, having the following amino acid sequence:

H ₂ N-Asp ¹ -Asn ² -Trp ³ -Trp ⁴ -Pro ⁵ -Lys ⁶ -Pro ⁷ -Pro ⁸ -His ⁹ -Gln ^l0 -Gly ¹¹ -Pro ^l2 -Arg ^l3 -Pro ^l4 -Pro ^l5 -Arg ^l6 -Pro ¹⁷ -Arg ¹⁸ -Pro ¹⁹ -Lys ²⁰ -Pro ²¹ -COX, where X is a OH group (azemiopsin, AF-3) or NH ₂ (azemiopsin amide, AF-3a).

The claimed peptide interacts with muscle efficiency with high efficiency, as was shown in tests for the inhibition of the binding of radioactive alpha-bungarotoxin with membrane preparations of the electric organ of the stingray Torpedo californica containing muscle-type NHR. The half effect of a decrease in the binding of the radioactive toxin IC ₅₀ is 9.3 ± 0.6 nM. At the same time, in the case of neural NHR alpha7 type, this value is 22 ± 2 μM. Additionally, the claimed peptide completely blocks the conduction of a nerve impulse in a classical experiment on a neuromuscular preparation of a frog. In addition, the claimed peptide in a direct toxicological experiment in mice is 10 times less toxic than paralytic d-tubocurarine, which is used as a muscle relaxant in medicine. So, the dose that causes the death of half of the animals, LD ₅₀ for azemiopsin is 2.57 mg / kg, while for d-tubocurarine this value is 0.2 mg / kg.

The claimed peptide azemiopsin contains 21 amino acid residues, i.e. is a relatively small molecule, which simplifies its preparation by peptide synthesis. In addition, the peptide is linear and does not contain cysteine residues, which also simplifies its production by chemical synthesis or biotechnology, since the risk of formation of inactive products due to incorrect closure of disulfide bonds is eliminated.

The azemiopsin peptide AF-3 is obtained from a natural source - the poison of the Burmese viper Azemiops feae, which belongs to the family Viperidae, class Reptilia, order Sqamata, type Chordata.

The peptide azemiopsin amide AF-3a is obtained by chemical synthesis, it differs from the natural peptide by the presence of an amide group at the C-terminal proline residue and exhibits similar biological properties.

The inventive peptide is a new compound and is not a close analogue of any peptides or proteins. The invention is illustrated in figures 1-5.

Figure 1. Separation of viper venom Azemiops feae by gel filtration. For separation, a Superdex 75 column (1 × 30 cm), an elution rate of 30 ml / h and 0.1 M ammonium acetate buffer (pH 6.2) were used.

Figure 2. Isolation of the azemiopsin peptide (AF-3) by reverse phase chromatography. For separation, a Jupiter C 18 reverse phase column (4.6 × 250 mm) and an elution rate of 1 ml / min were used. The separation is carried out in a linear gradient of acetonitrile concentration in the presence of 0.1% trifluoroacetic acid. Detection at a wavelength of 226 nm.

Figure 3. Azemiopsin inhibition of the binding of radioactive alpha-bungarotoxin to Torpedo californica electrical organ membranes containing muscle-type nicotinic cholinergic receptors. C is the concentration of azemiopsin, mol / L.

Figure 4. Azemiopsin inhibition of binding of radioactive alpha-bungarotoxin to GH4C1 cells heterologously expressing alpha7 type nicotinic cholinergic receptors. C is the concentration of azemiopsin, mol / L.

Figure 5. Dependence of the number of dead animals on the dose of azemiopsin administered.

The invention is illustrated by examples.

Example 1. Isolation of azemiopsin from the venom of a Burmese viper Azemiops feae.

At the first stage, the whole venom of the viper Azemiops feae is subjected to separation by gel filtration on a Superdex 75 column (Figure 1). The collected fractions were lyophilized and fraction VI marked with a line (FIG. 1) was subjected to further separation by reverse phase chromatography on a Jupiter C18 column (4.6 × 250 mm). Fractionation is carried out in a linear gradient of the concentration of acetonitrile from 2 to 62% (v / v) in 0.1% trifluoroacetic acid for 60 minutes at a speed of 1 ml / min. Detection is carried out by optical absorption at 226 nm. The AF-3 fraction (Figure 2) contains the azemiopsin peptide, which is used for further studies.

Example 2. Establishment of the amino acid sequence of azemiopsin.

Determination of the N-terminal amino acid sequence of the peptide is carried out by stepwise degradation according to Edman on a Procise 492 automated sequencer (Applied Biosystems, USA). As a result, the amino acid sequence of 10 N-terminal amino acid residues is established:

NH2-Asp ¹ -Asn ² -Trp ³ -Trp ⁴ -Pro ⁵ -Lys ⁶ -Pro ⁷ -Pro ⁸ -His ⁹ -Gln ^l0 .

The complete amino acid sequence was determined by MALDI mass spectrometry using an Ultrafex II TOF / TOF time-of-flight mass spectrometer (Bruker Daltonik GmbH, Germany). Fragment ion spectra were generated by laser-induced dissociation accelerated by low-energy shock dissociation using helium as an auxiliary gas. As a result of the analysis of fragment ions, the complete amino acid sequence of the azemiopsin AF-3 peptide is established:

NH2-Asp ¹ -Asn ² -Trp ³ -Trp ⁴ -Pro ⁵ -Lys ⁶ -Pro ⁷ -Pro ⁸ -His ⁹ -Gln ^l0 -Gly ^ll -Pro ^l2 -Arg ^l3 -Pro ^l4 -Pro ^l5 -Arg ¹⁶ - Pro ¹⁷ -Arg ¹⁸ -Pro ¹⁹ -Lys ²⁰ -Pro ²¹ -OH.

Example 3. Chemical synthesis of azemiopsin amide AF-3a.

Joining the first amino acid. Preparation of Fmoc-Pro-P (Fmoc = 9-fluorenylmethyloxycarbonyl).

200 mg of a 2-chlorotrityl chloride polymer with a hydroxyl content of 1.0 mmol / g was washed with anhydrous methylene chloride. 374 mg (1.1 mmol) of Fmoc-Pro-OH and 374 μl (2.2 mmol) of diisopropylethylamine are dissolved in 5 ml of methylene chloride, the solution is stirred for 5 minutes and then added to the polymer. The reaction is carried out for 1 hour at room temperature and with stirring. At the end of the reaction, the polymer is filtered off and washed three times successively with methylene chloride, ethyl alcohol, dimethylformamide.

Description of one synthetic polypeptide chain extension cycle.

Obtaining Fmoc-Lys (Boc) -Pro-P:

a) The peptidyl polymer obtained in the previous step is treated with a 20% solution of piperidine in dimethylformamide for 20 minutes. The polymer is washed successively with 5 ml of the following solvents: dimethylformamide - 3 times for 2 minutes, a mixture of dioxane-water (2: 1) - 2 times for 5 minutes, dimethylformamide - 5 times for 2 minutes.

b) 505 mg (1.1 mmol) of Fmoc-Lys (Boc) -OH, 150 mg (1.1 mmol) of 1-hydroxybenzotriazole and 172 μl (1.1 mmol) of N, N'- are dissolved in 5 ml of dimethylformamide diisopropylcarbodiimide, the solution is stirred for 10 minutes at 0 ° C and then poured onto the suspended polymer. The reaction is carried out for 4 hours with periodic stirring. At the end of the reaction, the polymer is filtered off, washed with dimethylformamide and treated with 5 ml of a mixture of Ac ₂ O-pyridine-dimethylformamide (20:20:60) for 1 h, after which the polymer is washed successively with dimethylformamide, isopropanol, dimethylformamide.

The synthesis of the polypeptide chain is carried out manually in a glass flow reactor (2 × 20 cm) according to the following protocol for each synthetic cycle (based on 8-10 ml of solvent per 400 mg of the starting polymer); during the condensation reaction (step 6), the volume of the reaction mixture 5 -7 ml:

1. DMF (dimethylformamide) (5 × 2 min);

2. 20% piperidine in DMF (20 min);

3. DMF (3 × 2 min);

4. dioxane-water, 2: 1, (2 × 5 min);

5. DMF (5 × 2 min);

6. Condensation reaction: 5 molar equivalents of activated Fmoc amino acids (4 hours);

7. DMF (3 × 2 min);

8. acylation: Ac ₂ O-pyridine-dimethylformamide, 20:20:60, (1 h);

9. DMF (3 × 2 min);

10. isopropanol (3 × 2 min.);

To activate Fmoc-derived amino acids using a DIPCDI / HOBT (N, N'-diisopropylcarbodiimide / 1-hydroxybenzotriazole) mixture to a solution of 1.1 mmol (5 equivalents) of an Fmoc-protected amino acid and 150 mg (1.1 mmol, 5 equivalents) HOBt in 4 ml of DMF was added 170 μl (1.1 mmol, 5 equivalents) of DIPCDI, the solution was stirred for 10 minutes.

The completeness of the condensation reaction is monitored using the ninhydrin or, in the case of the N-terminal proline, isatin tests after step 6 of the synthetic protocol.

The following amino acid derivatives are used for synthesis: Fmoc-Gly-OH, Fmoc-Lys (Boc) -OH, Fmoc-His (Trt) -OH, Fmoc-Pro-OH, Fmoc-Gln (Trt) -OH, Fmoc-Asn ( Trt) -OH, Fmoc-Asp (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Arg (Pbf) -OH.

Cleavage of the peptide from the polymer.

For the reaction of cleavage of the peptide from the polymer and simultaneous release of the protective groups of the amino acid side chains, 800 mg of the peptidyl polymer are selected. 15 ml of a mixture of TFA (trifluoroacetic acid) - H ₂ O in a volume ratio of 97.5: 2.5 are added to the peptidyl polymer, the suspension is stirred for 2 hours, then the resulting peptide solution is filtered off from the polymer, washed with 5 ml of TFA and the excess TFA is evaporated under reduced pressure . The peptide is precipitated with 100 ml of ethyl ether, filtered and washed with ether (5 × 20 ml). The precipitate was dissolved in 5 ml of 10% acetic acid for 20 minutes, filtered and washed with 5 ml of 10% acetic acid. The resulting peptide solution was lyophilized and desalted on a column (2.5 × 60 cm) with Sephadex G-10 in 0.1 M acetic acid. The peptide was purified using reverse phase HPLC in an acetonitrile gradient (from 10% to 35% in 75 min.) In 0.1% TFA at an eluent flow rate of 3 ml / min; the absorption of the eluate was recorded at a wavelength of 226 nm. Fractions corresponding to the main peak in the chromatogram are collected and lyophilized. The molecular weight of the peptide determined by mass spectrometry was 2539.1 Da, and the theoretical calculated molecular weight was 2539.4 Da.

Example 4. The biological activity of azemiopsin.

The biological activity of azemiopsin is determined in experiments on competition with radioactive alpha-bungarotoxin for binding to nicotinic cholinergic receptors of the muscle or alpha7 type. Torpedo californica stingray electric organ membranes and GH4C1 cells heterologously expressing the human alpha7 receptor are used as a source of muscle and alpha7 cholinergic receptors. For competitive binding experiments, a suspension of the membranes of an electric organ (1.2 nM alpha-bungarotoxin-binding sites) or GH4C1 cells (0.4 nM alpha-bungarotoxin-binding sites) in 20 mM Tris-HCl buffer, pH 8.0, containing bovine serum albumin at a concentration of 1 mg / ml, incubated for 90 min with various concentrations of azemiopsin. Then add radioactive [ ¹²⁵ I] -labeled alpha-bungarotoxin at a concentration of 0.2-0.4 nM and incubate for another 5 minutes. To determine nonspecific binding, the preparations are incubated with alpha-cobratoxin at a concentration of 1 μM before adding radioactive bungarotoxin. After incubation with radioactive bungarotoxin, the suspension of membranes or cells is filtered through GF / F glass fiber filters (Whatman, England), previously incubated in a solution of 0.25% polyethyleneimine. Then the filters are washed three times with 3 ml of 20 mM Tris-HCl buffer, pH 8.0, containing bovine serum albumin at a concentration of 0.1 mg / ml. The amount of bound radioactivity was determined on a 1470 Automatic Gamma Counter (Perkin Elmer) instrument.

Example 5. Determination of acute toxicity of azemiopsin

Semi-lethal dose of LD ₅₀ , i.e. the average dose of azemiopsin causing the death of half of the members of the test group of animals was determined in a direct toxicological experiment. Male mice of strain BALB / c weighing 18-22 g were randomly divided into 6 groups, 5 animals each. After preliminary weighing of the mice, a solution of the peptide in sterile water was administered intraperitoneally at a concentration of 2 to 10 mg / kg. The death of mice occurred after 10-30 minutes. The total volume of the injected solution was 230 μl. As a control, an intraperitoneal injection of 230 μl of sterile water was used. The results are shown in table 1.

table 1 Determination of LD _{50 of} azemiopsin. Group number Dose of azemiopsin (mg / kg) The number of dead mice number % one 10 5 one hundred 2 8 5 one hundred 3 four four 80 four 2.7 3 60 5 2 one twenty 6 0 0 0

To calculate the semi-lethal dose, a graph was built of the dependence of the number of dead animals on the amount of the drug administered, and on its basis the LD ₅₀ value was determined, which amounted to 2.57 ± 0.27 mg / kg

Example 6. The study of the action of azemiopsin on the neuromuscular preparation of a frog. The azemiopsin peptide is dissolved in Ringer's physiological solution: NaCl - 0.65%, KCl - 0.014%, CaCL ₂ - 0.012%, NaHCO ₃ - 0.020%. A frog neuromuscular preparation is prepared. Then apply a solution of the peptide (1 mg / ml) for 20 minutes at the junction of the sciatic nerve and calf muscle. With further electrical irritation of the sciatic nerve, the calf muscle does not contract, that is, a complete blockade of the nerve impulse is observed. As a control, a similar neuromuscular preparation was used, incubated for 20 min in Ringer's solution, for which muscle contraction was observed with electrical irritation of the sciatic nerve.

Claims (2)

1. The azemiopsin peptide selectively interacting with muscle type nicotinic cholinergic receptors and having the following amino acid sequence:
NH ₂ -Asp ^l -Asn ² -Trp ³ -Trp ⁴ -Pro ⁵ -Lys ⁶ -Pro ⁷ -Pro ⁸ -His ⁹ -Gln ¹⁰ -Gly ¹¹ -Pro ^l2 -Arg ¹³ -Pro ^l4 -Pro ^l5 -Arg ^l6 -Pro ^l7 -Arg ^l8 -Pro ^l9 -Lys ²⁰ -Pro ²¹ -COX,
where X is an OH or NH ₂ group. 2. The use of the peptide according to claim 1 as a muscle relaxant of peripheral action in surgery, dermatology and cosmetology.

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