RU2521657C1 - Analgesic peptide from sea anemone urticina grebelnyi - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: analgesic peptide has the following amino acid sequence: H₂N-Ile¹-Ser²-Ile³-Asp⁴-Pro⁵-Pro⁶-Cys⁷-Arg⁸-Phe⁹-Cys¹⁰-Tyr^ll-His^l2-Arg¹³-Asp^l4-Gly^l5-Ser^l6-Gly^l7-Asn^l8-Cys¹⁹-Val²⁰-Tyr²¹-Asp²²-Ala²³-Tyr²⁴-Gly²⁵-Cys²⁶-Gly²⁷-Ala²⁸-Val²⁹-COOH. Said peptide can be used as a medicinal agent for treating pathologies associated with tissue acidosis and treating neurologic diseases associated with ASIC3 receptor function, as well as for determining the topology and molecular mechanisms of the function of the ASIC3 channel, and in test systems for detecting and testing novel anti-pain preparations, agonists and antagonists of the ASIC3 receptor.

EFFECT: analgesic action owing to inhibition of the functional activity of proton-activated ion channels.

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Description

The invention relates to biochemistry, specifically to biologically active peptides with analgesic or anti-inflammatory effects, which may find application in medicine or scientific research.

Currently, one of the main reasons for people seeking medical help is pain. Pathological pain that occurs in various conditions is an undesirable phenomenon that dramatically reduces the quality of life, performance and causing suffering. Up to 40% of adults in developed countries suffer from chronic pain. The most widely used analgesic drugs are traditionally used: morphine (and other opioids), aspirin (as well as other non-steroidal anti-inflammatory drugs) and a variety of non-specific substances (anticonvulsants, antidepressants). Despite the large assortment of analgesic agents of various chemical nature, some types of pain conditions, such as various neuropathies, are practically insensitive to these agents. In addition, the non-specificity of the action of the above substances in some cases causes undesirable side effects, which greatly limits the possibility of their use.

Recently, special attention has been paid to the development and production of fundamentally new analgesic agents that specifically act on the molecular mechanisms of pain with minimal side effects, which implies the use of highly selective agents that can specifically turn on, turn off or modify a specific fragment along the path of the pain signal from the lesion to the brain.

An important role in many pathological processes is played by the family of proton-sensitive ion channels (ASICs), which include various combinations of ASICla, ASIClb, ASIC2a and ASIC3 subunits [Krishtal O. The ASICs: signaling molecules? Modulators? // Trends Neurosci. 2003. V.26. P.477-483]. ASIC channels are expressed by many types of nerve cells, mainly peripheral and sensory neurons of the central nervous system (CNS), including neurons sensitive to pain stimuli. These channels perform an important function of signal transduction when pH changes in the intercellular space. Most types of ASIC channels are responsible for the sensation of pain, accompanied by tissue acidosis in muscle, cardiac, cerebral ischemia, epilepsy, various inflammatory and infectious processes, corneal damage and other pathologies [Ugawa S., Ueda T., Ishida Y., Nishigaki M., Shibata Y., Shimada S. Amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors // J. Clin. Invest. 2002. V.110, P.1185-1190]. It has been established that one of the key roles in the perception of high-intensity pain stimuli in H ⁺ -induced pathological processes is played by ASIC3 channels present in sensitive neurons of the peripheral nervous system [Deval E., Noel J., Lay N., Alloui A., Diochot S. , Friend V., Jodar M., Lazdunski M., Lingueglia E. ASIC3, a sensor of acidic and primary inflammatory pain // Embo J. 2008. V.27, P.3047-3055; Deval E., Gasull X., Noel J., Salinas M., Baron A., Diochot S., Lingueglia E. Acid-sensing ion channels (ASICs): pharmacology and implication in pain // Pharmacol. Ther. 2010. V.128, P.549-558; Deval E., Noel J., Gasull X., Delaunay A., Alloui A., Friend V., Eschalier A., Lazdunski M., Lingueglia E. Acid-sensing ion channels in postoperative pain // J. Neurosci. 2011. V.31, P.6059-6066].

Currently, the set of active ASIC3 channel modulator ligands used in practice is limited mainly by amiloride and non-steroidal anti-inflammatory drugs acting as inhibitors [Voilley N., de Weille J., Mamet J., Lazdunski M. Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors // J. Neurosci. 2001. V.21, P.8026-8033], and mammalian neuropeptides NPRF and NPSF activating ASIC3 channels [Lingueglia E., Deval E., Lazdunski M. FMRFamide-gated sodium channel and ASIC channels: a new class of ionotropic receptors for FMRFamide and related peptides // Peptides. 2006. V.27, P.1138-52].

So far, among the selective compounds, four active components are known that interact with ASIC channels. First of all, this is the polypeptide from the tarantula venom Psalmopoeus cambridgei, Psalmotoxin 1 (PcTx1) [Escoubas P., De Weille JR, Lecoq A., Diochot S., Waldmann R., Champigny G., Moinier D., Menez A., Lazdunski M. Isolation of a tarantula toxin specific for a class of proton-gated Na ⁺ channels // J. Biol. Chem. 2000. V.275, P.25116-25121], which is a highly specific and potent inhibitor of homomeric channels of the ASIC1a type [Chen X., Kalbacher H., Grunder S. The Tarantula Toxin Psalmotoxin 1 Inhibits Acid-sensing Ion Channel (ASIC) 1a by Increasing Its Apparent H ⁺ Affinity // J. Gen. Physiol. 2005. V.126. P.71-79].

The second substance is a polypeptide from the sea anemone Anthopleura elegantissima, called APETx2. It selectively inhibits the homomeric ASIC3 channels and, with less selectivity, the heteromeric ASIC1a + 3, ASIC1b + 3 and ASIC2b + 3 channels [Deval E., Gasull X., Noel J., Salinas M., Baron A., Diochot S., Lingueglia E. Acid-sensing ion channels (ASICs): pharmacology and implication in pain // Pharmacol. Ther. 2010. V.128, P.549-558; Deval E, Noel J., Gasull X., Delaunay A., Alloui A., Friend V., Eschalier A., Lazdunski M., Lingueglia E. Acid-sensing ion channels in postoperative pain // J. Neurosci. 2011. V.31, P.6059-6066].

The third and fourth polypeptide inhibitor, mambalgins (mambalgin-1 and mambalgin-2), consist of 57 a.a. and have in their composition 8 cysteines, which form 4 disulfide bonds between themselves. They were isolated from the venom of the snake Dendroaspis polylepis polylepis [Diochot, S., Baron, A., Salinas, M., Douguet, D., Scarzello, S., Dabert-Gay, AS, Debayle, D., Friend, V. , Alloui, A., Lazdunski, M. and Lingueglia, E. Black mamba venom peptides target acid-sensing ion channels to abolish pain // Nature. 2012. V.490, P.552-555]. Polypeptides differ from each other by one a.a. (Tyr4 in mambalgin-1 and Phe4 in mambalgin-2). Mambalgins are capable of reversibly inhibiting the recombinant homomeric ASIC1a, heteromeric ASIC1a + ASIC2a and ASIC1a + ASIC2b, as well as ASIC1b and ASIC1a + ASIC1b channels with IC ₅₀ values of 55 nM, 246 nM, 61 nM, 192 nM and 72 nM, respectively.

Despite the fact that ASIC1a is also partially involved in the perception of inflammatory processes, the modification of its conductivity using various pharmaceutical agents from a practical point of view has a significantly lower therapeutic potential compared with the search and implementation of pharmaceutical agents on ASIC3. Therefore, the introduction of Psalmotoxin 1 and mambalgins may not be as effective. In addition, the disadvantage of mambalgins is a large number of disulfide bonds, which complicates the correct folding of synthetic analogues of these peptides after synthesis, and therefore reduces the efficiency of their preparation.

The disadvantage of APETx2 is its incomplete inhibitory effect on ASIC3 receptors. It is known that this channel has a complex dynamics of operation, which is why in the electrophysiological experiments the common current passing through this receptor is divided into a fast and long (stationary) component. Fast current has a large amplitude and lasts less than 3 seconds. A continuous current with a not so large amplitude and a slow rate of increase ultimately exceeds the fast current in the total number of cations passed through the membrane. The selectivity of APETx2 is such that the polypeptide can inhibit only the fast component of the total current of the ASIC3 channel; therefore, the biological effect of reducing a smaller part of the current through ASIC3 receptors is not so significant. A greater analgesic effect can be expected from compounds that effectively reduce both components of the current.

The invention solves the problem of expanding the arsenal of new-generation analgesic and anti-inflammatory drugs that have a targeted effect on the cellular target, namely the ASIC3 proton-controlled ion channel, which is involved in the perception, processing and transmission of signals associated with changes in the pH of the extracellular environment in the peripheral nervous system .

The inventive peptide agent, called Ugr 9-1 (full name according to the classification of π-AnmTX Ugr 9a-1), has a pronounced inhibitory effect on the fast component and on the continuous component of the current through the ASIC3 channel (Fig.6 and 7).

The inventive peptide can be used as an analgesic agent to alleviate pain conditions caused by the participation of ASIC3 channels in the pathological processes of transmission of painful proton-induced stimuli, primarily associated with tissue acidosis, as well as to clarify the topology and molecular mechanisms of ASIC3 functioning.

The problem is solved due to the structure of the peptide Ugr 9-1, having the following amino acid sequence:

H ₂ N-Ile ^l -Ser ² -Ile ³ -Asp ⁴ -Pro ⁵ -Pro ⁶ -Cys ⁷ -Arg ⁸ -Phe ⁹ -Cys ¹⁰ -Tyr ^ll -His ^l2 -Arg ¹³ -Asp ¹⁴ -Gly ^l5 -Ser ^l6 -Gly ^l7 -Asn ^l8 -Cys ¹⁹ -Val ²⁰ -Tyr ²¹ -Asp ²² -Ala ²³ -Tyr ²⁴ -Gly ²⁵ -Cys ²⁶ -Gly ²⁷ -Ala ²⁸ -Val ²⁹ -COOH

Ugr 9-1 is a compound of polypeptide nature, its amino acid sequence contains 29 amino acid residues, and its structure is stabilized by 2 disulfide bonds, which makes the molecule, on the one hand, extremely stable and resistant to degradation, and on the other hand, reduces the cost of its synthesis . And as a result of this, it improves the consumer properties of drugs based on it.

The inventive analgesic peptide can be isolated by chromatographic methods from nematocysts of marine anemones, synthesized by chemical methods from individual amino acids, or obtained as a recombinant protein by genetic engineering methods. Precursor protein structure of the inventive polypeptide (1) obtained on the basis of a nucleotide sequence of messenger RNA, having the following structure: agtttgtaactgcaggctatcgctcaaccggaatcagaagcggaataatcttcaacaaagaaaATGGAAAGGATTTTGCTGTGCTTCATCGTGGCTACTTTGGTAGCCATTTCAATGGCCAACCCAAGGCCTATCAGCATCGATCCCCCTTGTTGGACTTGTTATTACCGAGACAGCAGTGGTAACTGTGCGTTCGACGCCATCGCCTGTGGAGGAGCGCGCAAGAGAGTTCCCAAGCCTATCAGCATCGATCCCCCTTGTAGGACTTGTTATTACCGAGACAGCAGTGGTAACTGTGTGTACGACGCCTTCGGCTGTGGAGGAGCGCGCAAGAGAGTTCCCAAGCCTATCAGCATCGATCCCCCTTGTAGGACTTGTTATTACCGAGACAGCAGTGGTAACTGTGTGTACGACGCCTTCGGCTGTGGAGGAGCGCGCAAGAGAGTTCCCAAGCCTATCAGCATCGATCCCCCTT GTAGGTTTTGTTATCACCGAGACGGCAGTGGTAACTGTGTGTACGACGCCTACGGCTGTGGAGCAGTGCGCAAGTAAtcctgtcaaaaactcataatataactgtgatgaccgctaatacatttgcttaaaatttactaaataaaatcattactcccaaaaaaaaaaaaaaaa

The resulting gene encodes, in an open reading frame, 162 amino acid residues and a stop codon, where a signal peptide of 19 amino acid residues precedes the structure of a gap containing four peptides between which short cleavable fragments are located. Among the four peptides obtained after limited proteolysis of one protein, two have the same amino acid sequence. The inventive analgesic peptide is located in the C-terminal part of the gap, it is synthesized in a single copy (per gap), and no post-translational modifications in its composition are found.

The inventive peptide exhibits analgesic activity in mammals, which was shown in tests on mice of the CD-1 line, both for the model of thermal hypersensitivity after the introduction of Freund's adjuvant (Fig. 8) and for the model of stimulation of vinegar cramps (Fig. 9). In in vitro experiments, the claimed peptide is able to exhibit a pronounced inhibitory effect on the fast component and on the continuous component of the current through the ASIC3 channels of a person expressed in frog oocytes (Figs. 6 and 7).

The invention is illustrated by figures.

FIG. 1. The structure of the nucleotide sequence of the gene encoding the analgesic peptide Ugr 9-1 and the amino acid sequence of its precursor protein. The signal peptide sequence is shown in bold, mature peptides obtained by maturation of the precursor protein are underlined, the sequence of the analgesic peptide corresponding to that previously obtained by the automatic method of sequencing the amino acid sequence is highlighted in a frame.

FIG. 2. Comparison of the amino acid sequence of Ugr 9-1 and related structural homologs. Amino acid residues that differ from the Ugr 9-1 sequence are indicated in black. A gray highlight shows the location of cysteine amino acid residues characteristic of most of the analyzed peptides, and a diagram of their closure to disulfide bonds is shown below.

FIG. 3. The stages of chromatographic isolation of the analgesic peptide Ugr 9-1. A. Elution profile from a TSK 2000SW gel filtration column (7.5 × 600 mm, 125 Å, 10 μm; Toyo Soda Manufacturing Co., Japan) equilibrated with 10% acetonitrile in the presence of 0.1% TFA at a flow rate of 0.5 ml / min B. RP-HPLC on a Jupiter C ₅ column (4.6 × 250 mm, 300 Å, 5 μm) (Phenomenex, USA) in the presence of 0.1% TFA, an elution rate of 1 ml / min and a linear gradient of acetonitrile concentration. B. RP-HPLC on a Vydac C ₁₈ column (4.6 × 250 mm) (Grace, United States) in the presence of 0.1% TFA, an elution rate of 1 ml / min and a linear gradient of acetonitrile concentration. The elution time of active fractions at each stage is indicated by a black rectangle.

FIG. 4. Recombinant Ugr 9-1 Recombinant Peptide Gene Cloning Scheme. The insertion site of the amino acid residue Met for subsequent cleavage with cyanogen bromide is indicated by a black triangle, black rectangles indicate the domain of thioredoxin and a sequence of 6 histidines for metal affinity chromatography.

FIG. 5. HPLC of the hydrolyzate of 14 mg of the chimeric protein on a Jupier Cs reverse phase column (Phenomenex, USA) 10 × 250 mm. Separation at a rate of 5 ml / min was carried out in a linear gradient of acetonitrile concentration in the presence of 0.1% TFA. Detection at a wavelength of 210 nm. Elution time Ugr 9-1 is highlighted in gray.

FIG. 6. Measurement of the inhibitory activity of Ugr 9-1 on the fast current component of ASIC3. A. Recordings of measured currents across the membrane, carried out on frog X. laevis oocytes expressing human ASIC3 channels. The measured cells were fixed at a potential of 50 mV, the flow rate of about 1 ml / min. The activation of the fast channel component was caused by a rapid change in the pH of the buffer solution from 7.8 to 5.5. Curves of induced currents without the addition of a peptide are given (control) and after preliminary incubation of an oocyte with a peptide at concentrations of 5, 10, 20, and 40 μM. B. A dose-response curve constructed as the ratio of current after preincubation of Ugr 9-1 with an oocyte to the excitation current level of a control experiment.

FIG. 7. Measurement of the inhibitory activity of Ugr 9-1 on the continuous current component of ASIC3. A. Recordings of measured currents across the membrane, carried out on frog X. laevis oocytes expressing human ASIC3 channels. The measured cells were fixed at a potential of -50 mV, the flow rate of about 1 ml / min. Activation of the continuous channel component was caused by a rapid change in the pH of the buffer solution from 7.3 to 4.0. Curves of induced currents are given without adding a peptide (control) and when a peptide is introduced into an external solution at concentrations of 1, 5, 20, and 50 μM. B. The dose-response curve constructed as the ratio of the current measured in the presence of Ugr 9-1 to the level of the excitation current of the control experiment.

FIG. 8. The analgesic effect of Ugr 9-1 in the test of thermal hypersensitivity caused by PAF. The measured paw withdrawal time (n = 7) for control animals and animals after injection of Ugr 9-1 at doses of 0.5, 0.1 and 0.01 mg / kg is shown. Statistically significant differences were noted from the group “nat. p-p ”** - p <0.005, * - p <0.05.

FIG. 9. The analgesic effect of Ugr 9-1 in the acid pain stimulation test (“vinegar cramps”). The measured number of seizures (n = 10) for control animals and animals after injection of Ugr 9-1 at doses of 0.5, 0.1 and 0.01 mg / kg is given. Statistically significant differences were noted from the group “nat. p-p ”** - p <0.005, * - p <0.05.

The invention is illustrated by examples.

Example 1. Isolation of the analgesic peptide Ugr 9-1

Marine anemones of the species Urticina grebelnyi are kept in cold seawater at a temperature of 4 ° C, removed and placed in a clean flat dish to collect poison from special stinging cells of nematocysts located throughout the body. The animals are initially washed with distilled water to remove impurities, then, using a sterile loop, they irritate surface cells by repeating pressures with medium pressure. The secretion is washed off with distilled water containing proteolytic enzyme inhibitors 1 mM phenylmethylsulfonyl fluoride (PMSF) and 5 mM ethylenediaminetetraacetic acid (EDTA) in a clean dish. The secretion is concentrated on a rotary evaporator and lyophilized for subsequent storage at -20 ° C. Animals are placed in cold sea water for rehabilitation and feeding, after which the new procedure for re-sampling the poison can be repeated after two weeks.

Isolation and purification of the analgesic peptide is carried out according to the developed scheme (Figure 3), including gel filtration, which allows to remove high molecular weight impurities (stage 1), and two stages of reverse phase (RP) HPLC on Jupiter C ₅ and Vydac C ₁₈ columns (stage 2 and 3). Detection of chromatographic fractions at all stages of isolation is carried out spectrophotometrically at wavelengths of 210 and 280 nm. In the separation, volatile solutions are used: for gel filtration, 10% acetonitrile and 0.1% trifluoroacetic acid (TFA); for RP-HPLC, water with 0.1% TFA and acetonitrile with 0.1% TFA. At stages 2 and 3, separation is carried out in a linear gradient of acetonitrile concentration from 0 to 50% in 50 minutes and from 0 to 15% in 5 minutes, then from 15 to 40% in 50 minutes, respectively.

The activity of the intermediate fractions is confirmed in biological tests on animals or by electrophysiology methods on human ASIC3 channels expressed in frog oocytes.

Example 2. The establishment of a partial amino acid sequence

Determination of the N-terminal amino acid sequence of the purified peptide is carried out by the method of stepwise degradation according to Edman on a Precise 492 automatic sequencer (Applied Biosystems, USA). As a result, the amino acid sequence of the first 25 amino acid residues is established:

H ₂ N-Ile ^l -Ser ² -Ile ³ -Asp ⁴ -Pro ⁵ -Pro ⁶ -Cys ⁷ -Arg ⁸ -Phe ⁹ -Cys ¹⁰ -Tyr ¹¹ -His ¹² -Arg ¹³ -Asp ^l4 -Gly ^l5 -Ser ^l6 -Gly ^l7 -Asn ^l8 -Cys ¹⁹ -Val ²⁰ -Tyr ²¹ -Asp ²² -Ala ²³ -Tyr ²⁴ -Gly ²⁵ -

Example 3. The establishment of the structure of the gene and the complete amino acid sequence of Ugr 9-1

To obtain the total mRNA fraction, use a freshly prepared sample of sea anemone (50 mg), homogenize with pestle in 0.5 ml RNAwiz RNA isolation buffer (Ambion, Canada), incubate at room temperature for 5 minutes, then add 0.1 ml of chloroform, shake and incubate for another 10 minutes . The resulting mixture was centrifuged at 4 ° C (15 min, 14000 g) and the upper aqueous phase was selected. 0.25 ml of distilled water and 0.5 ml of isopropanol are successively added, centrifuged at 4 ° C (15 min, 14000 g). 1 ml of 75% ethanol was added to the precipitate and precipitated by centrifugation at 4 ° C (5 min, 14000 g). The precipitate of RNA is dried in air and dissolved in 50 μl of distilled water that does not contain RNase. The concentration of RNA is determined spectrophotometrically.

To synthesize the first cDNA strand, 5 μg of total RNA was mixed with the Sar (T) 20 primer: (AAG CAG TGG TAA CAA CGC AGA GTA C (T) 30N-1N, where N = mixture A, C, G, T; and N -1 = mixture A, G, C), bring the volume of the mixture to 12 μl with distilled water, incubate for 5 min at 70 ° C and cool in an ice bath. Add 4 μl of a 5-fold reaction buffer (250 mM Tris-HCl (pH 8.3 at 25 ° C), 250 mM KCl, 20 mM MgCl ₂ , 50 mM dithiothreitol), 1 μl RNAsin ribonuclease inhibitor (Promega), 2 μl 10 mM dNTP Incubated for 5 minutes at 37 ° C. The reaction is carried out for 1 hour at a temperature of 42 ° C, adding 1 μl (200 activity units) of RevertAid ™ reverse transcriptase to the reaction mixture (Fermentas, Latvia).

To determine the complete nucleotide sequence of a gene encoding a protein precursor of the Ugr 9-1 peptide, one synthesizes: one universal T7cap primer (GTA ATA CGA CTC ACT ATA GGG CAA GCA GTG GTA ACA ACG CAG AGT); two degenerate primers calculated from the partial amino acid sequence of the desired protein to determine the 3'-terminal sequence of Ug1 (ATS TOT ATS GAT CCN CCN CCN TGY MG), Ug2 (CCA CCC TGT AGN TTY TGY TAY CA); and two specific primers for determining the 5'-terminal sequence of Ug3 (CGG TCA TCA CAG TTA TAT TAT GAG), Ug4 (ATT ATG AGT TTT TGA CAG GAT TAC).

PCR was performed using Taq polymerase (Eurogen) under the following conditions of 94 ° C for 20 sec, 55 ° C for 20 sec, 72 ° C 60 sec, 35 cycles. Amplified fragments of about 150 bp (in an experiment to determine the 3'-terminal sequence) or about 600 bp (in an experiment to determine the 5'-terminal sequence) clone into the plasmid pAL-TA (Eurogen). The nucleotide sequence of several clones is determined on a multi-channel automatic sequencer PRISM 3100-Avant (ABI-Perkin Elmer). The full-sized structure of the gene encoding the protein precursor is determined after multiple alignment of all obtained sequences (Figure 1).

The correctness of the final amino acid sequence is confirmed by comparing the calculated and measured molecular weights of the peptide. The uniqueness of the active peptide sequence is confirmed by comparison with a database of known amino acid sequences (Figure 2).

Example 4. Determination of relative molecular weight

The identity of the purified peptide is confirmed by mass spectrometric analysis. Mass spectra were obtained on an Ultraflex II TOF / TOF MALDI time-of-flight mass spectrometer (Broker Daltonik, Germany), with identification of positive ions in a reflex mode. The matrix used is α-cyano-4-hydroxycinnamic acid (10 mg / ml) in 50% (v / v) acetonitrile containing 0.1% (v / v) TFA. To calibrate the device using a standard mixture of peptides with a molecular weight range of 700-3500 Da (Sigma, USA).

The measured average molecular weight of the natural Ugr 9-1 polypeptide is 3135.1 Da. The calculated average molecular weight differs from that measured by 0.35 Da. Given the accuracy of the mass determination method, it is concluded that there is no post-translational modification of this peptide.

Example 5. The creation of a genetic engineering construct to obtain recombinant Ugr 9-1

To obtain a genetic engineering construct capable of expressing Ugr 9-1 in E. coli cells, using the bioinformatics methods, the amino acid sequence is converted to the nucleotide sequence taking into account the optimization of the use of codons in the host cell (E. coli). 3 oligonucleotide primers are synthesized that overlap the complete amino acid sequence of the protein and contain the methionine residue and restriction enzyme sites BgIII and Xhol: F1 (GAA TTA GAT CTC ATG ATT TCC ATT GAT CCG CCG TGC CGT TTT TGC TAT CAT); Rl (CGC ATC ATA CAC GCA ATT GCC GGA GCC ATC ACG ATG ATA GCA AAA ACG GCA); R2 (GGA TTC CTC GAG STA CAC CGC GCC GCA GCC ATA CGC PBX ATA CAC GCA ATT). With primers F1 and R1, the first round of PCR is performed. The resulting amplified DNA is used as a template for the second round of PCR, which is carried out with a pair of primers, F1 and R2. Both rounds of PCR were performed under similar conditions of 94 ° C for 20 sec, 55 ° C for 20 sec, 72 ° C for 20 sec, 25 cycles using Taq polymerase (Eurogen). (Gibco).

The Ugr 9-1 sequence is inserted into the fusion protein after the thioredoxin fragment and the sequence of 6 amino acid residues of histidine for prokaryotic expression as a water-soluble chimeric protein (Figure 4). For this, after treatment with restriction enzymes EcoRI and Xhol, the assembled Ugr 9-1 gene and expression vector pET32b + (Novagen) are ligated with each other. The result is a plasmid encoding a fusion protein under the control of the T7 promoter. The resulting plasmid is transformed into XL1-Blue cells and the correct assembly and ligation by sequencing is checked.

Example 6. Obtaining recombinant Ugr 9-1

BL21 (DE3) cells are transformed with the expression vector pET32 + Ugr 9-1. After selection on antibiotic plates (ampicillin), the cells are seeded in a 200 ml flask and grown to an optical density of ~ 0.6 with stirring and aeration. Expression induction is carried out by adding isopropyl-β-D-1-thiogalactopyranoside to a concentration of 0.2 mM, after which the cells are grown for another 12 hours at 24 ° C. Cells are pelleted and resuspended in disintegration buffer (20 mM Tris-HCl pH 7.2, 150 mM NaCl). Disintegrate cells with ultrasound, precipitate cell debris and inclusion bodies by centrifugation. The target fusion protein is contained in the soluble fraction.

As a result of the expression of the created construct, the production of recombinant Ugr 9-1 occurs as part of a fusion protein with thioredoxin, where the sequence of thioredoxin together with polyhistidine strand precedes the active peptide. Metal affinity chromatography on a Co ²⁺ absorbent is carried out in a buffer (20 mM Tris-HCl pH 7.2, 150 mM NaCl), a buffer (150 mM imidazole, 300 mM NaCl, 20 mM Tris, pH 7.5) is used to elute the chimeric protein. The collected protein was desalted on PD-10 columns (GE Healthcare) using distilled water as eluent and lyophilized.

The dried chimeric protein is dissolved in 0.1 M HCl to a protein concentration of 2 mg / ml, digested with bromine cyan for 15 hours in the dark, using a 600-fold molar excess of cyanogen bromide to the chimeric protein. The solvent and excess bromine cyan are removed in a vacuum concentrator, then the mixture of limited proteolysis products is dissolved in chromatographic buffer A (0.1% TFA) and applied to a 10 × 250 mm Jupier C ₅ (Phenomenex) reverse phase column to isolate the desired polypeptide (Figure 5) . Fractionation is carried out in a linear gradient of acetonitrile concentration from 0 to 60% (v / v) in 0.1% (v / v) TFA for 60 minutes with an elution rate of 5 ml / min. Detection is carried out by optical absorption at 210 nm.

The purity and accuracy of the synthesis of recombinant Ugr 9-1 are checked using mass spectrometric analysis, a comparison of the chromatographic mobilities of the natural and recombinant polypeptide on a Luna C ₁₈ (Phenomenex) 2 × 150 mm analytical column, and a comparison of biological properties.

Example 7. Expression of ASIC3 channels in frog oocytes

RNA encoding the ASIC3 channel is obtained using the RiboMAX Large Scale RNA Production System (Promega) reagent kit, for which 20 μl of a 5-fold transcription buffer is added to 33 μl of the cleaved and purified plasmid containing the T7 promoter in front of the coding region of the human ASIC3 gene ( 400 mM HEPES-KOH, 120 mM MgCl ₂ , 10 mM spermidine, 200 mM dithiothriethol), 20 μl of a mixture of ribonucleoside triphosphates (25 mm ATP, CTP, UTP and 2 mm GTP), 7 μl Cap-analogue (at a concentration of 40 mm) and 10 μl of T7 RNA polymerase (Promega). The reaction mixture was incubated for 3 hours at 37 ° C. RNA purification is carried out using the phenol / chloroform method. The resulting samples were dissolved in water, analyzed in agarose gel electrophoresis and stored at -80 ° C for several weeks.

ASIC3 channels are obtained as a result of their expression in the membranes of oocytes of the frog Xenopus laevis. For this, female oocytes isolated from the ovary are treated with type I or type II collagenase (Sigma-Aldrich, USA) at a concentration of 1 mg / ml for 2 hours to remove the follicular membrane. Defolliculated oocytes are placed in sterile ND96 medium (NaCl 96 mM, KCl 2 mM, CaCl ₂ 1.8 mM, MgCl ₂ 1 mM, HEPES 5 mM titrated NaOH to pH 7.8) and incubated overnight at a temperature of 15-16 ° C. Injection of 2.5-10 ng mRNA of the human ASIC3 channel (AJ272063) is performed under a MBS-10 binocular microscope (Russia) using the Eppendorf5242 microinjector (Germany). After injection, the oocytes are stored for 2-3 days at a temperature of 19 ° C, and then up to 7 days at a temperature of 15 ° C in ND-96 medium, which is pre-titrated with NaOH to pH 7.4 and to which an antibiotic gentamicin is additionally added to a concentration of 50 μg / ml and pyruvate to a concentration of 5 mm.

Measurement of ion currents through the ASIC3 channels is carried out at a frequency of 100 Hz by two-electrode fixation of the membrane potential at a level of -50 mV using a GeneClamp 500 amplifier (Axon Instruments, USA) in a working chamber with a free volume of 45 μl. The data is filtered at a frequency of 20 Hz and digitized using the L780 ADC (LCard, Russia) using home-made programs. Microelectrodes are filled with 3 M KCl solution.

Example 8. The effect of Ugr 9-1 on the fast component of currents ASIC3

A two-electrode membrane potential fixation system is used to measure channel conductivity. A potential difference of -50 mV is supported by a GeneClamp 500 digital amplifier (Axon Instruments, CA), signals are recorded at a frequency of 100 Hz. Microelectrodes are filled with a solution of 3 M KCl. Buffer solution used Ca ²⁺ -free ND-96 (96 mM NaCl, 2 mM KCl, 0.1 mM BaCl _2, 1 mM MgCl _2, HEPES 5 mM NaOH titrated to the desired pH value)

The current is measured in the laminar flow of an ND96 solution (pH 7.8) at a rate of 1 ml / min. ASIC3 channels are activated by a sharp change in pH from 7.8 to 5.5 due to the rapid replacement of the working solution in the chamber with a solution with a pH value of 5.5, in which 5 mM MES is used as a buffer instead of 5 mM HEPES. The pulse length of a change in pH is 1 second. To reduce nonspecific binding to the channel, weighed portions of test samples are dissolved in buffer solutions containing 0.1% BSA. The application of the test sample is started 5 seconds before the activation buffer is fed into the measuring chamber. In total, 7 different concentrations of the peptide are used in at least three repetitions, for which the amplitude of the fast component of the current through ASIC3 is measured (Fig. 6). The percentage of inhibition of the observed currents is calculated as the ratio of the peak amplitude of the current during the application of the toxin to the average amplitude of the peak of the control currents before and after the application of the toxin. Inhibition is reversible, which is manifested in the complete restoration of the amplitude caused by acidification of the currents through the ASIC3 channels after complete washing of Ugr 9-1. For the fast current components, 100% inhibition is observed at a concentration of 40 μM.

Based on the results of measurements of all concentrations, a curve is constructed of the% current of the fast component relative to the control relative to the concentration of the peptide. Based on this curve, the 50% inhibitory concentration IC ₅₀ 10 ± 0.6 μM and the Hill coefficient 1.86 ± 0.19 are determined.

Example 9. The effect of Ugr 9-1 on the continuous component of the currents ASIC3

A two-electrode membrane potential fixation system is used to measure channel conductivity. A potential difference of -50 mV is supported by a GeneClamp 500 digital amplifier (Axon Instruments, CA), signals are recorded at a frequency of 100 Hz. Microelectrodes are filled with a solution of 3 M KCl. The current is measured in a laminar flow of an ND96 solution (96 mM NaCl, 2 mM KCl, 0.1 mM BaCl ₂ , 1 mM MgCl ₂ , HEPES 5 mM titrated NaOH to pH 7.3) at a rate of 1 ml / min. ASIC3 channels are activated by a sharp change in pH from 7.3 to 4.0 due to the rapid replacement of the working solution in the chamber with a solution with a pH value of 4.0, in which 10 mM acetic acid is used instead of 5 mM HEPES as a buffer. The pulse length of the change in pH is 3 seconds. To reduce nonspecific binding to the channel, weighed portions of test samples are dissolved in buffer solutions containing 0.1% BSA. The application of the test sample is started 3 seconds before the activation buffer is fed into the measuring chamber and continued for the duration of the activation solution supply. In total, 7 different concentrations of the peptide are used in at least three repetitions, for which the amplitude of the continuous component of the current through ASIC3 is measured, which reaches its maximum value 3-4 seconds after the activation of the channels (Fig. 7). The percentage of inhibition of the observed currents is calculated as the ratio of the maximum current amplitude during the application of the toxin to the average maximum amplitude of the control currents before and after the application of the toxin. Inhibition is reversible, which is manifested in the complete restoration of the amplitude caused by acidification of the currents through the ASIC3 channels after the complete washing of the sample. For the continuous current component, no more than 48 ± 2% inhibition is observed at a concentration of 50 μM or higher.

According to the results of measurements of all concentrations, a curve is constructed of the% current of the continuous component relative to the control to the Ugr 9-1 concentration. Based on this curve, the 50% inhibitory concentration IC ₅₀ 1.44 ± 0.19 μM and Hill coefficient 1.48 ± 0.27 are determined.

Example 10. Testing the analgesic activity of Ugr 9-1 in the test of thermal hypersensitivity

Tests are carried out on male CD-1 white mice weighing 20-30 g. Mice are divided into 5 groups of 7 in each. Inflammation in mice, in addition to the control group, is caused by introducing into the hind paw pad an inflammatory agent, which is 20 μl of a mixture of complete Freund's adjuvant (PAPF solution 1: 1 (v / v). For the control group of animals (Fig. 8, the group without PAP) is administered only 20 μl of physiological saline: After 24 hours, 200 μl of physiological saline is administered intravenously to animals without PAF and physical pp groups, the remaining groups are injected with 200 μl of test samples in sterile physiological saline at a dose of 0.5, 0.1 and 0.01 mg / kg . Measured e is carried out 30 minutes after intravenous administration. Record the paw withdrawal latency time, subjected to the action of an inflammatory agent, from the hot plate (t = 53 ° C).

The results are processed statistically, the significance of differences in the results of the control and experimental groups is determined using ANOVA and Tukey test. The analgesic effect is measured by the increase in the time elapsed from the moment the animal was placed on the plate until the inflamed paw was withdrawn (Fig. 8).

Example 11. Testing the analgesic activity of Ugr 9-1 in an acid pain stimulation test.

Tests are carried out on male CD-1 white mice weighing 20-30 g. Mice are divided into 4 groups of 10 each. The peptide is dissolved in sterile physiological saline and 100 μl of the solution are administered intraperitoneally 30 minutes before the introduction of the acetic acid solution. Three doses of 0.5, 0.1 and 0.01 mg / kg are used. For the control group of animals, just 100 μl of physiological saline is administered (Fig. 9). The analgesic effect is determined based on the count of writhing caused by intraperitoneal injection of 100 μl of a solution of 1% acetic acid for 15 minutes of observation. Cramps are a specific pain reaction accompanied by characteristic movements of animals, which include contractions of the abdominal muscles, alternating with their relaxation, extension of the hind limbs and curving of the back.

The results are processed statistically, the significance of differences in the results of the control and experimental groups is determined using ANOVA and Tukey test. The analgesic effect is measured by a decrease in pain sensitivity to visceral and inflammatory types of pain, which is expressed in a decrease in the number of cramps over a measured period of time.

Claims (1)

An analgesic peptide from a sea anemone Urticina grebelnyi having the following amino acid sequence:
H ₂ N-Ile ^l -Ser ² -Ile ³ -Asp ⁴ -Pro ⁵ -Pro ⁶ -Cys ⁷ -Arg ⁸ -Phe ⁹ -Cys ¹⁰ -Tyr ¹¹ -His ^l2 -Arg ¹³ -Asp ^l4 -Gly ^l5 -Ser ^l6 -Gly ^l7 -Asn ^l8 -Cys ¹⁹ -Val ²⁰ -Tyr ²¹ -Asp ²² -Ala ²³ -Tyr ²⁴ -Gly ²⁵ -Cys ²⁶ -Gly ²⁷ -Ala ²⁸ -Val ²⁹ -COOH

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