RU2422459C1 - Peptide purinoceptor modulator - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly to a biologically active peptide. The peptide exhibits a modulating action on end-organs involved in nerve signal generation. The given peptide has an amino acid sequence: Gly¹-Tyr²-Cys³-Ala⁴-Glu⁵-Lys⁶-Gly⁷-lle⁸-Arg⁹-Cys¹⁰-Asp¹¹-Asp¹²-Ile¹³-His¹⁴-Cis¹⁶-Thr¹⁷-Gly¹⁸-Leu¹⁹-Lys²⁰-Cys²¹-Lys²²-Cys²³-Asn²⁴-Ala²⁵-Ser²⁶-Gly²⁷-Tyr²⁸-Asn²⁹-Cys³⁰-Val³¹-Cys³²-Arg³³-Lys³⁴-Lys³⁵.

EFFECT: invention can be used in medicine for studying the pain mechanisms, and also for detection and testing of new P2X3 receptor modulators.

7 dwg, 1 tbl, 7 ex

Description

The invention relates to biochemistry, specifically to biologically active polypeptides that have a modulating effect on cellular receptors involved in the generation of a pain signal and can find application in medicine and scientific research.

According to modern concepts, membrane proteins that cause signal transmission (various receptors and ion channels) play a crucial role in the life of cells. Disruption of the receptor systems can be the cause of diseases and pathologies. One of the urgent problems of modern physiology, bioorganic chemistry, and related sciences is the directed regulation of the function of cell receptors / ion channels by selective action on these membrane proteins. Peptide toxins contained in natural poisons and capable of specifically affecting potential-dependent sodium, calcium, potassium channels as well as mechano-, thermo-, and chemo-excitable channels, which belong to a large group of so-called pain receptors, are a powerful tool for structural and functional studies, as well as the basis for creating new effective painkillers [Lewis RJ, Garcia ML Therapeutic potential of venom peptides // Nat. Rev. Drug Discov., 2003, V.2, P.790-802].

Currently, a number of components of natural poisons are known that have an analgesic effect, many of them are at the stage of clinical or preclinical trials. So, analgesic activity is possessed by: α-conotoxin Vc1.1, acting on neuronal nicotinic acetylcholine receptors [dark R.J. et al. The synthesis, structural characterization, and receptor specificity of the alpha-conotoxin Vc1.1 // J. Biol. Chem., 2006, Vol. 281, P.23254-23263; US 7,348,400]; ω-conotoxins MVIIA (SNX-111) and CVID (AM-336), which are calcium channel blockers [Olivera B.M. et al. Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus venom // Biochemistry, 1987, Vol.26, P.2086-2090; Nielsen K.J. et al. Structure-activity relationships of omega-conotoxins at N-type voltage-sensitive calcium channels // J. Mol. Recognit., 2000, Vol.13, P.55-70; Scott D.A. et al. Actions of intrathecal omega-conotoxins CVID, GVIA, MVIIA, and morphine in acute and neuropathic pain in the rat // Eur. J. Pharmacol., 2002, V.451, P.279-286; US 7,101,849]; χ-conopeptide MrIA-noradrenaline transporter inhibitor [Bryan-Lluka L.J. et al. chi-Conopeptide MrIA partially overlaps desipramine and cocaine binding sites on the human norepinephrine transporter // J. Biol. Chem., 2003, Vol. 278, P.40324-40329; US 7,326,682]; contulakin G - neurotensin receptor agonist [Craig A.G. et al. Contulakin-G, an O-glycosylated invertebrate neurotensin // J. Biol. Chem., 1999, Vol.274, P.13752-13759; US 6,696,408]; ρ conopeptide TIA antagonist of α-adrenergic receptors [Sharpe I.A. et al. Allosteric alpha 1-adrenoreceptor antagonism by the conopeptide rho-TIA // J. Biol. Chem., 2003, Vol. 278, P. 34451-34457; US 6,849,601]. For practical use, for example, the above-mentioned ω-conotoxin MVIIA (Ziconotide, Prialt) [Miljanich G.P. Ziconotide: neuronal calcium channel blocker for treating severe chronic pain // Curr. Med. Chem., 2004, V.11, P.3029-3040; US 5,859,186].

The disadvantages of these funds are:

(1) the introduction of drugs such as Prialt into the body is carried out by injection into the spinal column, which in many cases is difficult and requires the use of special medical equipment (for example, implanted pumps) and the work of highly qualified personnel;

(2) the analgesic activity of a number of substances may be accompanied by side effects due to the participation of target receptors in many physiological processes;

(3) some of these polypeptides contain oxidatively methionine residues, which leads to a decrease in the therapeutic potential of the corresponding drugs.

Ionotropic purinergic receptors (P2X) are ligand-controlled ion channels activated by extracellular adenosine triphosphate (ATP), some other nucleotides and their derivatives. P2X receptors are widespread in the human body. So, they are characteristic of peripheral neurons innervating various organs and tissues [Surprenant A., North R.A. Signaling at purinergic P2X receptors // Annu. Rev. Physiol., 2009, Vol. 71, P.333-359]. The potential importance of these targets for the development of medications is due to the ubiquitous presence of their endogenous ATP ligand in the body and the variety of conditions in which it can realize its activating effect. ATP is a coenzyme that is involved in many important reactions and is a source of energy for various processes, such as muscle contraction and protein biosynthesis. At the same time, numerous studies have shown that ATP released from damaged or inflamed tissues activates P2X receptors and initiates pain signals. The participation of P2X receptors in pain sensitivity associated with injuries, tumors, inflammation, migraines, and respiratory diseases is assumed. In humans, seven isoforms of P2X receptors are known, among which isoforms of P2X2, P2X3, P2X4 and P2X7 are believed to be involved in the pain perception system [Donnelly-Roberts D. et al. Painful purinergic receptors // J. Pharmacol. Exp. Ther., 2008, Vol.324, P.409-415]. P2X3 receptors are important and well-studied “participants” in pain processes in both the central and peripheral nervous systems, which allows us to consider them as a promising target in the treatment of pain conditions, and their antagonists as potential analgesic agents [Wirkner K. et al. P2X3 receptor involvement in pain states // Mol. Neurobiol., 2007, Vol. 36, P. 165-183].

Specific and selective P2X3 receptor inhibitors or modulators are currently poorly understood. Some low molecular weight P2X3 receptor antagonists are known, for example, 2 ′, 3′-O- (2,4,6-trinitrophenyl) -ATP [Virginio C. et al. Trinitrophenyl-substituted nucleotides are potent antagonists selective for P2X1, P2X3, and heteromeric P2X2 / 3 receptors // Mol. Pharmacol., 1998, Vol. 53, P. 969-973], other derivatives of mono- and dinucleotides [Ford K.K. et al. The P2X3 antagonist PI, P5-di [inosine-5 '] pentaphosphate binds to the desensitized state of the receptor in rat dorsal root ganglion neurons // J. Pharmacol. Exp. Ther., 2005, V. 315, P. 405-413; US 6,881,725], analogues of suramin [Hausmann R. et al. The suramin analog 4,4 ′, 4 ′ ′, 4 ′ ′ ′ - (carbonylbis (imino-5, l, 3-benzenetriylbis (carbonylimimino))) tetra-kis-benzenesulfonic acid (NF110) potently blocks P2X3 receptors: subtype selectivity is determined by location of sulfonic acid groups // Mol. Pharmacol., 2006, V.69, P.2058-2067], spinorfin [Jung K.Y. et al. Structure-activity relationship studies of spinorphin as a potent and selective human P2X3 receptor antagonist // J. Med. Chem, 2007, V.50, P. 4543-4547], A-317491 [Jarvis M.F. et al. A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2 / 3 receptors, reduces chronic inflammatory and neuropathic pain in the rat // Proc. Natl. Acad. Sci. U.S.A., 2002, V.99, P.17179-17184], derivatives of diaminopyrimidine [Carter D.S. et al. Identification and SAR of novel diaminopyrimidines. Part 1: The discovery of RO-4, a dual P2X3 / P2X2 / 3 antagonist for the treatment of pain // Bioorg. Med. Chem. Lett. 2009, Vol.19, P. 1628-1631; Jahangir A. et al. Identification and SAR of novel diaminopyrimidines. Part 2: The discovery of RO-51, a potent and selective, dual P2X3 / P2X2 / 3 antagonist for the treatment of pain // Bioorg. Med. Chem. Lett. 2009, Vol.19, P.1632-1635; US 7,589,090; US 7,531,547], tetrazole and arylamides [US 7,595,405], as well as piperazine and phenylthienopyrazole [Brotherton-Pleiss C.E. Discovery and optimization of RO-85, a novel drug-like, potent, and selective P2X3 receptor antagonist // Bioorg. Med. Chem. Lett, 2010, Vol. 20, 1031-1036; US 7,491,821].

The disadvantages of these substances are:

(1) the existence of targets other than purinergic receptors in some compounds;

(2) most do not have sufficient selectivity for homomeric P2X3 receptors;

(3) the low potential of many substances from the point of view of creating drugs due to non-optimal chemical structure (for example, the presence of many acid groups).

Known closest to the claimed compound RO-85 is a derivative of piperazine and phenylthienopyrazole [Brotherton-Pleiss C.E. Discovery and optimization of RO-85, a novel drug-like, potent, and selective P2X3 receptor antagonist // Bioorg. Med. Chem. Lett, 2010, Vol.20, 1031-1036]. A certain disadvantage of the known compound is its structure: RO-85 can only be obtained using multistage chemical synthesis.

The invention solves the problem of expanding the range of substances that specifically affect P2X3 receptors.

The problem is solved due to the structure of the compound PT1, modulating the activity of P2X3 purinergic receptors. PT1 is a polypeptide substance of nature and can be obtained by genetic engineering.

PT1 has the following amino acid sequence:

Gly ^l -Tyr ² -Cys ³ -Ala ⁴ -Glu ⁵ -Lys ⁶ -Gly ⁷ -Ile ⁸ -Arg ⁹ -Cys ^l0 -Asp ¹¹ -Asp ^l2 -Ile ^l3 -His ^l4 -Cys ¹⁵ -Cys ¹⁶ -Thr ^l7 -Gly ¹⁸ -Leu ¹⁹ -Lys ²⁰ -Cys ²¹ -Lys ²² -Cys ²³ -Asn ²⁴ -Ala ²⁵ -Ser ²⁶ -Gly ²⁷ -Tyr ²⁸ -Asn ²⁹ -Cys ³⁰ -Val ³¹ -Cys ³² -Arg ³³ -Lys ³⁴ -Lys ³⁵

The inventive peptide consists of 35 amino acid residues, can be isolated by chromatographic methods from the poison of the spider Geolycosa sp. or obtained using peptide synthesis, as well as genetic engineering methods.

The inventive peptide is able to selectively inhibit currents mediated by P2X3 purinergic receptors in rat sensory neurons, as well as under the conditions of expression of human P2X3 receptors in HEK 293 cells. It has been shown that, under the action of nanomolar concentrations of the peptide, the activating effect of the natural ATP agonist is significantly reduced, which is associated with - apparently, with the stabilization of the desensitized state of the receptors. The concentration of PT1, causing a 50% inhibition of currents mediated by P2X3 receptors in rat sensory neurons, (IC ₅₀ ) is 12 ± 2 nM with a Hill coefficient (n _H ) of 1.1 ± 0.2 (for RO-85 IC ₅₀ in rat recombinant receptor ratio is ~ 31 nM). Studies of the activity of PT1 in relation to a number of ion channels and ionotropic receptors confirm the specificity of the action of the peptide on P2X3 receptors.

PT1-based drugs may be effective in the treatment of pain of various etiologies. PT1 is the first polypeptide compound of nature capable of exerting a selective modulating effect on P2X3 receptors. The peptide is characterized by an extremely stable structure (contains the motive of the so-called "cystine node"), in addition, does not contain methionine residues. This is the basis for the assumption of a high potential of PT1 in terms of drug development.

The invention is illustrated by drawings.

Figure 1. Isolation of PT1 from Geolycosa sp. (A) Size exclusion chromatography of whole venom on a TSK 2000SW column (7.5 × 600 mm). The active fraction (*) was noted. (B) Reverse phase HPLC of fraction I on a Vydac 218TP54 C ₁₈ column (4.6 × 250 mm). The active fraction (*) was noted. (B) The third stage of purification of the active substance (repeated chromatography of fraction 1) using the same Vydac 218TP54 column. The active fraction corresponding to pure PT1 is marked (*).

Figure 2. Scheme for constructing an expression vector. Synthesis of the PT1 gene using oligonucleotides (table 1), indicated by arrows, includes ligation and PCR and is accompanied by cloning into the pET-32b vector. Below is shown a part of the obtained construct with functional elements: a promoter, a thioredoxin gene (Trx), a hexahistidine sequence (6His), a target PT1 gene, and a terminator.

Figure 3. Obtaining recombinant PT1. (A) Electrophoretic separation of samples obtained by isolation of a thioredoxin fusion protein with PT1 in SDS-PAGE (12%). (1, 6) - standards, the values of molecular masses (kDa) are given on the left; (2) the total cell lysate of E. coli BL21 (DE3) carrying the plasmid pET-32b with the PT1 gene insert before IPTG addition; (3) - after induction of expression of the target gene 0.2 mM IPTG; (4) - soluble protein fraction; (5) a fusion protein purified by affinity chromatography. (B) Separation of the products of the hydrolysis of the hybrid protein by enteropeptidase on a Jupiter C ₄ column (10 × 250 mm). The fraction corresponding to PT1 (*) was noted.

Figure 4. The effect of PT1 on currents mediated by P2X3 receptors in a culture of neurons from the ganglia of the dorsal roots of rat spinal cord. As an agonist, CTF is used at a concentration of 100 μM. (A) Recording currents in response to an agonist application when PT1 binds to P2X3 receptors in a closed state. Application of 10 nM PT1 leads to an increase in current amplitude (black curve) compared to the control (gray curve). (B) Current recording in the case when PT1 binds to P2X3 receptors in a desensitized state. Application of 10 nM PT1 leads to a decrease in the amplitude of the current (black curve) compared to the control (gray curve).

Figure 5. Investigation of the mechanism of action of PT1 on P2X3 receptors. The abscissa shows the time of application of the agonist from the beginning of the experiment. The ordinate shows the amplitude of the measured currents. Confidence intervals indicate the amplitude of the noise in each particular ion current record. The black horizontal line (PT1, 20 nM) shows the period of time during which the neuron was in solution with 20 nM PT1. An increase in the period between agonist applications against PT1 leads to a weakening of the blocking of the ion current mediated by P2X3 receptors. With a further increase in this period, blocking goes into potentiation.

6. The concentration dependence of the effect of PT1 on P2X3 receptors in the culture of neurons from the ganglia of the dorsal roots of the spinal cord of rats.

7. The effect of PT1 on recombinant human P2X3 receptors in HEK 293 (HER) cells and P2X3 rat neuromuscular ganglia (ZKG) neuron receptors. The diagram shows the relative decrease in the amplitude of the ion current caused by the application of CTF (100 μM) on P2X3 receptors in the presence of PT1 in an extracellular solution at a concentration of 20 nM and 50 nM. The white columns are the effect for rat ZKG neurons, the gray columns are for HEK 293 cells.

The invention is illustrated by the following examples.

Example 1. The selection of the peptide PT1

Whole venom spider Geolycosa sp. (SPP code- A267TDLS2-KZARNA) was provided by Fauna Research and Production Association LLP (Republic of Kazakhstan). A freeze-dried sample of 25 μl of the poison is dissolved in 0.1 ml of 150 mM NaCl, 30 mM NaH ₂ PO ₄ and applied to a TSK 2000SW gel filtration column (7.5 × 600 mm, 125 A, 10 μm; Beckman, USA) . Elution is carried out with the same solution at a rate of 0.5 ml / min. Detection is carried out by optical absorption of the eluate at a wavelength of 214 nm (figa). All fractions are assayed for activity against P2X3 receptors.

The active fraction I is then separated by reverse phase high performance liquid chromatography (HPLC) on a Vydac 218TP54 C ₁₈ column (4.6 × 250 mm, 300 Å, 5 μm; Separations Group, United States) in a linear gradient of acetonitrile concentration (0-15 % in 5 min, 15-45% in 50 min, 45-65% in 10 min) in 0.1% trifluoroacetic acid (TFA) at a flow rate of 1 ml / min (Fig. 1B). The individual active peptide PT1 is purified as a result of the third stage of separation of the active fraction 1 using reverse phase HPLC on the same column in a gentle gradient of acetonitrile concentration (0-15% in 5 min, 15-25% in 30 min) (Fig. 1B).

Example 2. Determination of the molecular weight of PT1

The identity of the purified substance is confirmed by mass spectrometric analysis. Use the desorption method of "soft" ionization - time-of-flight matrix-activated laser desorption / ionization (MALDI). Mass spectra were obtained on a MALDI Ultraflex II TOF / TOF mass spectrometer (Bruker Daltonik, Germany) with a time-of-flight mass analyzer; identification of positively charged ions was carried out in a reflex mode. The matrix used is α-cyano-4-hydroxycinnamic acid (10 mg / ml) in 50% acetonitrile containing 0.1% TFA. To calibrate the device using a standard mixture of peptides and proteins with a molecular weight range of 700-66000 Da (Sigma, USA). The measured monoisotopic molecular weight of natural PT1 is 3833.5 Da.

Example 3. The establishment of the amino acid sequence of PT1

The primary structure (amino acid sequence) of PT1 is established using a combination of Edman N-terminal sequencing, selective proteolysis and mass spectrometry. First, a reaction is carried out for the reduction of disulfide bonds and the alkylation of thiol groups. For this, 1 nmol of the isolated peptide is dissolved in 40 μl of a mixture: 0.5 M Tris-HCl (pH 8.5), 6 M guanidine hydrochloride, 2 mM EDTA. A 200-fold excess of dithiothreitol (5 M solution in acetonitrile) was added. The sample was incubated at 40 ° C for 4 hours, after which a 3-fold excess (with respect to dithiothreitol) of 4-vinylpyridine (50% solution in isopropanol) was added. The modification is carried out at room temperature for 15 minutes in the dark. The alkylated peptide was isolated by reverse phase HPLC on a Jupiter C ₅ column (2 × 150 mm, 300 Å, 5 μm; Phenomenex, USA). The alkylated reduced peptide has a mass of 4682.0 Da, which corresponds to eight attached 4-vinylpyridine molecules (molecular weight 105.1 Da). Thus, PT1 contains eight cysteine residues forming four intramolecular disulfide bonds. The amino acid sequence of S-pyridylethylated PT1 was determined by the Edman method on a Precise Model 492 automatic sequencer (Applied Biosystems, USA) in accordance with the manufacturer's procedure. The result is the complete amino acid sequence of PT1:

H ₂ N-Gly ^l -Tyr ² -Cys ³ -Ala ⁴ -Glu ⁵ -Lys ⁶ -Gly ⁷ -Ile ⁸ -Arg ⁹ -Cys ¹⁰ -Asp ¹¹ -Asp ¹² -Ile ¹³ -His ¹⁴ -Cys ¹⁵ -Cys ¹⁶ -Thr ¹⁷ -Gly ¹⁸ -Leu ¹⁹ -Lys ²⁰ -Cys ²¹ -Lys ²² -Cys ²³ -Asn ²⁴ -Ala ²⁵ -Ser ²⁶ -Gly ²⁷ -Tyr ²⁸ -Asn ²⁹ -Cys ³⁰ -Val ³¹ -Cys ³² - Arg ³³ -Lys ³⁴ -Lys ³⁵ -COOH

The calculated monoisotopic molecular weight of the peptide is 3833.6 Da, which differs from that measured by 0.1 Da. Thus, PT1 does not contain any modifications, with the exception of four disulfide bonds.

Example 4. Obtaining recombinant PT1

The amino acid sequence of PT1 is converted to its corresponding nucleotide sequence, taking into account the codon frequencies of Escherichia coli. Synthesize oligonucleotide fragments that overlap the entire sequence of the PT1 gene, while the “terminal” ones contain restriction sites for cloning into plasmid pET-32b (Novagen, USA; see table 1, FIG. 2).

The synthesis of the full-length sequence of PT1 is carried out using the method of ligation-polymerase chain reaction (PCR). F1 and F2 oligonucleotides (2 μM each), 1 mM ATP and 10 units are added to mixture 1. polynucleotide kinases (Promega, USA) in a ligation buffer solution (Promega, USA). Mixture 2 contains oligonucleotides F, R1, R2 (2 μM each) and 1 mM ATP in a ligation buffer. To carry out the phosphorylation reaction, mixture 1 is incubated in an incubator at 37 ° C for 30 minutes, then, to inactivate the enzyme, it is heated at 70 ° C for 20 minutes. 5 μl of mixtures 1 and 2 are combined, incubated for 15 min at 96 ° C, slowly cooled to room temperature, after which 0.5 units are added. Phage T4 DNA ligases (Promega, USA) and incubated for 18 h at 16 ° C. Then, PCR is performed using a 100-fold diluted ligase mixture with primers F and R. PCR is performed on a RTS-200 PCR amplifier (MJ Research, USA). The reaction mixture (200 μl) contains: PCR buffer solution (IBCh RAS), a mixture of four deoxynucleotides (0.2 mm each) (Promega, USA), primer oligonucleotides (20 pmol each), ~ 1 ng of template DNA and 2.5 units Taq polymerase (IBCh RAS). PCR conditions: denaturation (96 ° C) - 20 sec; annealing (50 ° С) - 20 sec; elongation (72 ° C) - 30 sec, 25 cycles.

Restriction of the PCR product and plasmid pET-32b is carried out in MultiCore buffer, which is optimal for the work of BamHI and KpnI enzymes (Promega, USA). To digest 1 μg of DNA, 4 units are used. each restrictase. The standard reaction mixture (20 μl) contains 2 μl of 10-fold restriction buffer and 2 μl of stock solution of each enzyme. The reaction is carried out for 2 hours at 37 ° C. As a control, restriction of the plasmid by each of the enzymes separately is carried out, the result is evaluated by the change in the mobility of the vector during agarose gel electrophoresis. The restriction of DNA and vector fragments was purified using agarose gel electrophoresis followed by DNA isolation from the gel using the Wizard SV Gel and PCR Clean-Up System reagent kit (Promega, USA) in accordance with the manufacturer's procedure.

After treatment with restriction enzymes KpnI and BamHI, the synthetic PT1 gene and the expression vector pET-32b are ligated to each other. The reaction mixture (15 μl) contains: 1.5 μl of a 10-fold ligation buffer, 10 mM ATP, 10 ng of a plasmid digested at the BamHI and KpnI sites, 10 ng of a PCR product digested at the same restriction sites, and 1 unit . Phage T4 DNA ligases. The ligase mixture is incubated for 18 hours at a temperature of 16 ° C in a water bath. The result is a plasmid encoding a chimera of a thioredoxin protein with PT1 under the control of the T7 promoter (figure 2).

The resulting mixture was transformed into E. coli cells of strain XL1 Blue using a Cellject electroporator (Eurogentec, Belgium). To carry out the transformation, 45 μl of a suspension of competent cells thawed on ice was placed in an electroporation cell and 1 μl of ligase mixture was added. The parameters of the electric pulse are set according to the recommendations of the manufacturer of the electroporator. After a pulse, 700 μl of ice-cold LB medium is immediately added to the cells, the suspension is incubated for 5 min on ice, and then 1 h at 37 ° C. The entire volume of the suspension was transferred to a Petri dish with solid LB medium with the addition of ampicillin (70 μg / ml) as a selective factor. The plates are incubated at 37 ° C for 18 hours. The E. coli XL1 Blue colonies obtained on the selective medium after the transformation are scattered by streaks on a new Petri dish with solid LB medium and ampicillin (70 μg / ml). The plates are incubated for 18 hours at 37 ° C. PCR is carried out with oligonucleotide primers F and R specific for the sequence of interest; total nucleic acid from the obtained cell mass is used as a matrix. Cellular material (~ 1 μl) is resuspended in the reaction mixture (20 μl), PCR is performed. Selected transformants are used to obtain an overnight liquid culture. A small amount of cellular material is transferred into 5 ml of LB liquid medium with the addition of ampicillin (70 μg / ml), the suspension is incubated for 18 h at 37 ° C. The preparative isolation of plasmid DNA is carried out using a set of reagents Wizard Plus SV Minipreps DNA Purification System (Promega, USA) in accordance with the methodology of the manufacturer.

The correct assembly and subsidy is checked by Sanger sequencing on a Model 373 DNA Sequencer (Applied Biosystems, USA) using ABI PRISM BigDye Terminator Cycle Sequencing Ready reaction kit reagents, AmpliTaq DNA polymerase, FS (Perkin Elmer, USA) and primer pET-HindSeq (5'-CTTCCTTTCGGGCTTTG-3 ′) in accordance with the recommendations of these manufacturers.

The production of the chimeric protein is carried out using the controlled expression of the corresponding gene in E. coli cells of strain BL21 (DE3). The overnight culture of cells transformed with the pET-320 expression vector containing the PT1 gene insert is diluted 200 times with LB liquid medium with the antibiotic ampicillin (100 μg / ml). Cells are incubated at 37 ° C and stirring (200 rpm) until an optical density of OD ₆₂₀ = 0.6 is reached, after which an inducer (isopropyl-β-D-1-thiogalactopyranoside, IPTG; 0.2 mM) is added to the culture and incubated at room temperature for 18 hours

Cells are pelleted by centrifugation (10 min, 4500 rpm) and resuspended in 25 ml of start affinity chromatography buffer (see below) with the addition of 1% Triton X-100. The destruction of cell structures is carried out using an ultrasonic disintegrator CPX 750 (Cole-Parmer Instruments, USA) with a gradual increase in amplitude from 25 to 35% in increments of 2%. Isolation of the fusion protein is carried out using affinity chromatography. The cell lysate is applied in the start buffer (50 mM Tris-HCl, pH 8, 300 mM NaCl) onto a 3 ml column with TALON Superflow Metal Affinity Resin resin (Clontech, USA) at a flow rate of 1 ml / min. To exclude the possibility of non-specific sorption, the column is washed with 50 mM Tris-HCl, pH 8, containing 500 mM NaCl, 5% glycerol (v / v), 1% Triton X-100 (v / v), 5 mM imidazole. For elution of the target protein using a buffer containing 50 mm Tris-HCl, pH 8, 300 mm NaCl, 150 mm imidazole. Detection is carried out by optical absorption of the eluate at 280 nm. The isolation and purification of the hybrid protein is controlled by electrophoresis of aliquots of the obtained fractions in a polyacrylamide gel (Fig. 3A).

The obtained fusion protein was subjected to desalination on a Jupiter C ₅ column (4.6 × 150 mm, 300 Å, 5 μm; Phenomenex, USA). Elution is carried out by a stepwise change in the concentration of acetonitrile (0-70%) in 0.1% TFA with a flow rate of 1 ml / min. The protein is dried in a vacuum concentrator, dissolved in 50 mm Tris-HCl, pH 8, the final protein concentration is ~ 1 mg / ml. A solution of the catalytic subunit of human enterokinase (IBCh RAS) is added to the protein solution at the rate of 1 unit. per 1 mg of protein, hydrolysis is carried out at room temperature for 18 hours. Separation of the products of the hydrolysis of the hybrid protein is carried out on a Jupiter C ₄ column (250 × 10 mm, 300 Å, 10 μm; Phenomenex, USA) in a linear gradient of acetonitrile concentration (0- 40% in 60 minutes, 40-80% in 20 minutes) in 0.1% TFA at a flow rate of 2 ml / min. Detection is carried out by optical absorption of the eluate at 210 and 280 nm (Fig.3B). The result is a recombinant PT1 yield of 3 mg with 1 liter of bacterial culture. The identity of recombinant and natural peptides is proved by mass spectrometry, N-terminal sequencing, comparison of chromatographic mobilities, as well as biological properties.

Example 5. Electrophysiological studies of the conductivity of P2X3 receptors in a culture of neurons from the ganglia of the dorsal roots of the spinal cord of rats

For the experiments, neurons are isolated from the ganglia of the dorsal roots of the spinal cord (posterior root ganglia) of 9-12 day old Wistar rats. The rats are quickly cut off their head, then the spinal canal is opened. Ganglia are located on the sides of the spinal canal in the intercostal space, which are carefully removed with tweezers and transferred to a Petri dish filled with extracellular solution (130 mM NaCl, 5 mM KCl, 2 mM CaCl ₂ , 2 mM MgCl, 20 mM HEPES, pH 7, 4) room temperature. After receiving a sufficient number of ganglia, they are transferred to a pre-prepared enzyme solution (1 mg of trypsin and 2 mg of collagenase in 2 ml of extracellular solution), heated to 35 ° C. Ganglia in the enzyme solution are incubated for 10 minutes. Then the ganglia are thoroughly washed with a clean (without enzymes) extracellular solution in another Petri dish. Single cells are isolated from the washed ganglia using thin needles. A single-cell cup is transferred to an electrophysiological setup. The experiments begin after all the cells settle to the bottom of the plate ~ 30 min after isolation.

Electrophysiological testing is carried out using the classical method of "patch clamp" in the configuration of voltage removal from the whole cell ("whole cell") using the approach of rapid application of a substance (agonist), which binds to P2X3 receptors and causes the opening of the ion channel. The electrophysiological installation consists of three parts: a microscope, a system for applying substances to the cell (“jumping table”; Pharma Robot, Ukraine) and a system for recording currents flowing through channels in the plasma membrane of the cell. A bouncing table is an essential element of the system of application of substances to the cell. Isolated neurons in a Petri dish are placed under a microscope with phase contrast on the setup. For the experiment, cells with a diameter of 8-12 μm are selected. A glass electrode-pipette filled with an intracellular solution (120 mM CsF, 10 mM Tris-HCl, pH 7.2) is fed to the selected cell; as a result, a tight contact is formed between the pipette and the cell membrane with a resistance of the order of GΩ. Then the cell on the pipette is lifted from the bottom of the cup and placed in the application tube. The solutions used in the experiment are in the chambers of the bouncing table. Four types of solutions are used in the experiment: solution No. 1 - the base extracellular solution in which the obtained cells are stored, solution No. 2 - the basic solution with the addition of an agonist in the required concentration, solution No. 3 - the basic solution with PT1 in the required concentration, solution No. 4 - stock solution with agonist and PT1 in the required concentrations. The application tube, in which the pipette with the test cell is located, is lowered into the chamber with the agonist solution. The camera is selected by turning the table. After lowering the application tube into the chamber, the valve opens, and under the influence of hydrostatic pressure, the solution is sucked into the application tube from the chamber of the bouncing table. During the absorption of the solution into the application tube (application of the solution), the ion current passing through the cell membrane is recorded. Immediately after this (in less than 2 seconds), the application tube moves into the chamber with the base extracellular solution. The solution from the chamber is drawn into the application tube and washes the cell. Thus, the “washing” of the cell from the agonist is carried out. The washing procedure is carried out at least 15 times over 3 minutes. 3 minutes after the application of the agonist, the procedure is repeated according to the same scheme. Three consecutive agonist applications that result in the registration of three identical ionic currents are considered statistically significant controls. After obtaining control currents, the test substance (PT1) is applied to the cell in different concentrations. Next, the agonist application procedure is repeated along with PT1, at which currents are also recorded. After applying the agonist, along with PT1, the washing of the cells from the agonist is also carried out against the background of PT1. The sequential application of an agonist with PT1 is stopped when the amplitude of the P2X3-mediated current remains unchanged. The ratio of the amplitude of the control current to the steady-state amplitude of the current against the background of a certain concentration of PT1 is an indicator of the effectiveness of the given concentration of PT1 and is plotted as a point on the dose-response graph.

Adding an agonist to an extracellular solution that washes a neuron (application of an agonist) causes an electrochemical response - the opening in the plasma membrane of a neuron of channels through which an ion current flows. Current is recorded at room temperature using a Model 2400 amplifier (AM Systems, USA). Electrodes for electrophysiological studies are made of borosilicate glass on a special P-97 Flamming / Brown installation (Sutter Instrument, USA); their resistance is 3-5 MΩ. Currents are recorded at a supported potential of -60 mV, which is close to the natural value of the resting potential of nerve cells. As agonists that cause P2X-mediated currents, adenosine triphosphate (ATP) and cytidine triphosphate (CTF) are used. A current with a fast rise phase (up to 12 ms) and a fast decline phase (up to 500 ms) corresponds to a current mediated by P2X3 receptors. The P2X3 receptor has three main conformational states: closed, open, insensitive (desensitized). In the absence of an agonist in the extracellular solution, the receptor is in a closed state. The application of an agonist causes the transition of the receptor from a closed state to an open state (activation). Further, the receptor passes from an open state to a state insensitive to an agonist (desensitization). Removal of the agonist from the extracellular solution leads to the transition of the complex from an insensitive state to a closed state (exit from desensitization). The activation time of the complex when using both agonists is from 2 to 4 ms. The time of complex desensitization using both agonists is 20-100 ms. The exit time of the complex from desensitization strongly depends on the agonist and is ~ 30 min for ATP and ~ 2 min for CTF. The effect of PT1 on each of the states is analyzed by selecting the appropriate protocol. When using CTF as an agonist, they act according to the protocol in which CTF is applied once every three minutes in a saturating concentration (100 μM).

The effect of PT1 depends on the state in which the receptor is located. The binding of PT1 to the receptor in the closed state is expressed in an increase in the amplitude of the current compared to the control (figa). The binding of PT1 to the complex in an insensitive state leads to a significant decrease in the current amplitude (Fig. 4B). Both effects are completely reversible. The decrease in current amplitude disappears with an increase in the period between agonist applications (Fig. 5). This suggests that PT1 slows the release of the receptor from desensitization. The experimental data are analyzed by the Hill equation. The concentration of PT1, at which the current amplitude is halved compared to the control value, (IC ₅₀ ) is 12 ± 2 nM, the Hill coefficient (n _H ) for the dependence of the magnitude of the PT1 effect on the concentration is 1.1 ± 0.2 ( 6).

Example 6. The effect of PT1 on the conductivity of P2X3 human receptors in the conditions of expression of their genes in HEK 293 cells

The human P2X3 gene (the original plasmid pReceiver-BO2a-P2X3, GeneCopoeia, USA) is cloned into the pIRES2-EGFP vector (Clonteth, USA) at the restriction sites EcoRI and ApaI. The restriction of plasmids pReceiver-BO2a-P2X3 and pIRES2-EGFP is carried out in MultiCore buffer. For splitting 1 μg of DNA, 2 units are used. restriction enzymes EcoRI and ApaI (Promega, USA). The standard reaction mixture (20 μl) contains 2 μl of a 10-fold restriction buffer and 1 μl of stock solution of each enzyme. The reaction is carried out for 1 h at 37 ° C. As a control, restriction of plasmids with each of the enzymes separately is carried out.

After restriction enzyme digestion, the P2X3 gene and pIRES2-EGFP vector are purified and ligated with each other. The reaction mixture (15 μl) contains: 1.5 μl of a 10-fold ligation buffer, 10 mM ATP, 10 ng of a plasmid digested at EcoRI and ApaI sites, 10 ng of a DNA fragment encoding P2X3 cleaved at the same restriction sites, and 1 unit . Phage T4 DNA ligases. The ligase mixture is incubated for 18 hours at a temperature of 16 ° C in a water bath. The selection of clones and isolation of the target plasmid is carried out similarly to the procedure described in example 4, using kanamycin (30 μg / ml) as a selective antibiotic. The ligation is checked by Sanger sequencing on a Model 373 DNA Sequencer using the procedure described in Example 4 using primers CF1 (5'-AGGCGTGTACGGTGGGA-3 '), P2X3-F2 (5'-CTGGCCGCTGCGTGAACTX-3 F3 (5′-CGTTTCTGAGAAAAGCAGCG-3 ′).

The obtained plasmid pIRES2-EGFP-hP2X3 was used to transfect HEK 293 cells. For this, cells were incubated in DMEM medium (GIBCO Invitrogen, USA) containing 10% fetal bovine serum at 37 ° C in an atmosphere of 5% CO ₂ . The day before transfection, the cells are transferred to a 24-well plate and incubated for 18 hours until about 60% of the cells are confluent. Transfection is carried out using lipofectamine (Invitrogene, USA). Two solutions are prepared for this: the first - 0.5 μg of the plasmid is dissolved in 25 μl of serum-free DMEM; the second - 2 μl of lipofectamine is dissolved in 25 μl of serum-free DMEM. Carefully add the lipofectamine solution to the DNA solution, mix, incubate for 30 min at room temperature, add 150 μl of serum-free DMEM. Change the medium in the cell wells to fresh DMEM without adding serum, add 200 μl of the diluted DNA-lipofectamine complex dropwise, mix gently, shaking the plate, incubate for 2 hours at 37 ° C in an atmosphere of 5% CO ₂ . Then the medium is changed to DMEM with the addition of serum, the cells are incubated at 37 ° C in an atmosphere of 5% CO ₂ for 1-2 days, after which they are used for conducting electrophysiological experiments. As a result, it is obtained that the observed effects upon exposure of PT1 to recombinant human P2X3 receptors in HEK 293 cells are similar to the effects of PT1 on P2X3 rat neuromuscular ganglia receptor neurons, but are less pronounced (Fig. 7).

Example 7. The selectivity of the PT1

In the membranes of neurons of the ganglia of the dorsal roots of the spinal cord, in addition to the P2X3 receptors, there are also P2X2 and P2X2 / 3 receptors [Burgard E.C. et al. P2X receptor-mediated ionic currents in dorsal root ganglion neurons // J. Neurophysiol., 1999, V. 82, P. 1590-1598]. To test the effect of PT1 on P2X2 and P2X2 / 3 receptors, use the protocol described in Example 5 with the following differences. Since the exit time from desensitization of P2X3 receptors is very long compared with P2X2 and P2X2 / 3 receptors, an agonist is applied with a period of 1 min. In this case, all the observed (except the first) responses are mediated only by P2X2 and P2X2 / 3 receptors. All currently known agonists of any of these receptors are not selective for only one type, however, the sensitivity of different types of receptors to different agonists is significantly different. While the sensitivity of the P2X2 / 3 receptor to ATP and α, β-methylene-ATP is approximately the same (Kd ~ 30 μM), the minimum concentration of α, β-methylene-ATP required to activate P2X2 receptors is at least 100 μM . Cell membranes may contain P2X2 and P2X2 / 3 receptors in different ratios. The equivalence of ion currents observed in response to the alternate application of ATP and α, β-methylene-ATP, indicates a significant predominance of P2X2 / 3 receptors in the cell membrane. Applying PT1 to such a cell, the effect of PT1 on P2X2 / 3 receptors is studied. In cells that generate ion currents of different amplitudes, larger in response to the application of ATP and very small in response to the application of α, β-methylene-ATP, the membrane contains mainly P2X2 receptors. The effect of PT1 on P2X2 receptors is studied on such cells. The result is that PT1 does not affect the ion currents mediated by P2X2 / 3 and P2X2 receptors.

In addition to P2X receptors, the membranes of the neuromuscular ganglia neurons also contain, for example, TRPV1 receptors. TRPV1 receptors have been shown to be involved in pain sensitivity [Caterina MJ et al. The capsaicin receptor: a heat-activated ion channel in the pain pathway // Nature, 1997, Vol. 389, P. 816-824]. The effect of PT1 on TRPV1 receptors is studied using the protocol described above, in which capsaicin at a concentration of 500 nM is used as an agonist; intracellular solution contains: 130 mM KCl, 10 mM HEPES, 20 mM BAPTA, 4 mM ATP, pH 7.2; extracellular solution contains: 130 mm NaCl, 5 mm KCl, 2 mm BaCl ₂ , 2 mm MgCl ₂ 20 mm HEPES, pH 7.4. The result is that PT1 does not affect ion currents mediated by activation of TRPV1 receptors.

Signal transmission in the nervous system occurs with the participation of voltage-dependent ion channels of various selectivities. The following protocol is used to study the effect of PT1 on voltage-gated ion channels. For sodium and potassium channels, to obtain ion currents with an amplitude close to maximum, the supported potential on the membrane is changed from -60 mV to +20 mV, for calcium - from -90 mV to 0 mV. The induced ionic current is measured in the base solution, and then in the base solution with the addition of PT1 of the studied concentration, the recorded currents are compared. The result is that PT1 does not affect ion currents mediated by the voltage-dependent ion channels of the posterior radicular ganglia neurons.

Claims (1)

Peptide PT1 modulating the activity of P2X3 purinergic receptors having the following amino acid sequence: Gly ¹ -Tyr ² -Cys ³ -Ala ⁴ -Glu ⁵ -Lys ⁶ -Gly ⁷ -Ile ⁸ -Arg ⁹ -Cys ¹⁰ -Asp ¹¹ -Asp ¹² - Ile ¹³ -His ¹⁴ -Cys ¹⁵ -Cys ¹⁶ -Thr ¹⁷ -Gly ¹⁸ -Leu ¹⁹ -Lys ²⁰ -Cys ²¹ -Lys ²² -Cys ²³ -Asn ²⁴ -Ala ²⁵ -Ser ²⁶ -Gly ²⁷ -Tyr ²⁸ -Asn ²⁹ -Cys ³⁰ -Val ³¹ -Cys ³² -Arg ³³ -Lys ³⁴ -Lys ³⁵ .

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