RU2629198C1 - Peptide with ability to inhibit migration of cells stimulated by tarc protein - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: peptide of the formula H-Gly-Ala-Ile-Pro-Leu-Arg-Lys-Leu-Lys-Thr-Trp-Tyr-OH with ability to inhibit cells migration stimulated with TARC chemokine and a method for its preparation are proposed.

EFFECT: new compounds have useful biological activity.

2 cl, 1 dwg, 2 ex

Description

The invention relates to biologically active peptides capable of inhibiting TARC chemokine-stimulated cell motility. A peptide of the formula H-Gly-Ala-Ile-Pro-Leu-Arg-Lys-Leu-Lys-Thr-Trp-Tyr-OH has been proposed. The claimed peptide can be used in immunology for the treatment of allergic diseases and in oncology to increase the effectiveness of anticancer therapy of leukemia.

Chemokines are a group of structurally related proteins of a small size (8-12 kDa). They are cytokines that induce the migration of leukocytes into the lymphatic organs and foci of inflammation that control the activation and differentiation of cells. Chemokines act through specific membrane receptors.

TARC - thymus and activation regulated chemokine (thymus and activation-regulated chemokine, or CCL17) refers to the subfamily CC or β-chemokines, in the structure of which two cysteine residues are located side by side (-c-c-) [1]. TARC specifically binds to the CCR4 receptor. The main population of CCR4 + cells is normally composed of T-helper cells, which provide a humoral immune response to antigens (type 2 T-helpers) [2-4].

Type 2 T-helpers are known to play an important role in the development of immune pathologies, such as allergies and autoimmune disorders [5]. A significant increase in the blood chemokine TARC was shown for atopic dermatitis [6, 7], asthma [8], and allergic rhinitis [9].

High expression of CCR4 receptor was detected in adult T-cell lymphoma / leukemia cells and skin T-cell lymphoma [10, 11]. It was shown that skin lesions characteristic of these neoplasias are due to CCR4-dependent migration of tumor cells into the dermis.

All these facts indicate the need for the development of therapeutic approaches based on the inhibition of the interaction of the CCR4 receptor with its ligand. Some steps in this direction have already been taken. Several chemical CCR4 antagonists of various structures were developed and monoclonal antibodies against the CCR4 receptor were obtained, which showed a rather high anti-inflammatory and antitumor activity in preclinical trials [12, 13]. However, the use of chemical antagonists in the clinic can be associated with problems such as: low efficiency, toxicity, difficulties in assessing the pharmacokinetic characteristics and metabolism.

In this regard, it seems appropriate to obtain a CCR4 receptor antagonist with minimal side effects. As such a substance, a peptide fragment of human TARC can be used that has TARC antagonistic properties.

An object of the present invention is to synthesize peptides devoid of adverse reactions that inhibit TARC-mediated migration of lymphoid cells.

The problem is solved by synthesis of a peptide of the formula: TARC 17-28 - H-Gly-Ala-Ile-Pro-Leu-Arg-Lys-Leu-Lys-Thr-Trp-Tyr-OH. The inventive peptide contains 12 amino acid residues. The synthesis of the claimed peptide is carried out by solid-state method using Fmoc technology, which is currently a routine task, taking into account the length of the peptide. Purification of the peptide is carried out by conventional single-stage high performance liquid chromatography (HPLC). All this makes obtaining the inventive antagonist much simpler and cheaper than obtaining "humanized" antibodies.

The non-obviousness of the invention is due to the fact that the task is solved by synthesizing a peptide of the sequence 17-28 TARC, although not a single fragment of this protein is known from the literature that can inhibit TARC-stimulated migration of lymphoid cells. Thus, it did not follow from the literature that the search for a peptide inhibitor of TARC-stimulated lymphoid cell motility among fragments of the molecule may lead to the desired results. The inventive peptide does not have immunogenic and toxic effects, since it is a low molecular weight fragment of the endogenous chemokine TARC.

The synthesis of the claimed peptide is carried out according to the standard technology of peptide synthesis on the solid phase using the most modern Fmoc- (9-fluorenylmethoxycarbonyl) technology [14]. A copolymer of styrene with 1% divinylbenzene with an acid labile anchor group was used as an insoluble carrier. The following protections were used to block the functional groups of the amino acid side chains: tert-butyl for the hydroxyl groups of threonine and tyrosine; tert-butyloxycarbonyl (Boc) - protection for the ε-amino group of lysine and the indole tryptophan ring; 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) - protection for guanidine function of arginine. The peptide chain was increased by one amino acid, starting synthesis from the C-terminus. To create amide bonds, 2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) was used in the presence of 2 equivalents of N-methylmorpholine.

Trifluoroacetic acid with scavengers was used for the final release and cleavage of the peptides from the carrier. The crude solid phase synthesis product was purified using preparative high performance liquid chromatography (HPLC).

Example 1. Synthesis of the dodecapeptide H-Gly-Ala-Ile-Pro-Leu-Arg-Lys-Leu-Lys-Thr-Trp-Tyr-OH, the corresponding sequence of 17-28 thymic-associated regulatory chemokine (TARC)

We used derivatives of L-amino acids from Bachem (Switzerland), TBTU, TIBS (triisobutylsilane) from Fluka (Switzerland). For the synthesis, dimethylacetamide, dichloromethane (DCM), 4-methylpiperidine and trifluoroacetic acid from Fluka (Switzerland) were used.

Analytical and preparative HPLC was performed on a Gilson instrument (France); acetonitrile from Panreac (Spain) was used.

Analytical HPLC was performed on a Diasfer 110-C18 column (Russia). The eluents used were: buffer A — 0.1% TFA, buffer B — 80% acetonitrile in buffer A, elution with a gradient of buffer B from 20 to 80% in 30 min at a rate of 1 ml / min; detection at 220 nm. Preparative HPLC was performed on a Diasorb C16 130T column (25 × 250 mm), particle size of the sorbent 10 μm. Buffer A — 0.1% TFA, buffer B — 80% acetonitrile in buffer A was used as eluents. Elution was performed with a 0.5% gradient per minute of buffer B in buffer A from 100% buffer A at a rate of 10 ml / min. Peptides were detected at a wavelength of 220 nm.

¹ H-NMR spectra were recorded on a WH-500 Bruker 500 MHz spectrometer (Germany) in DMSO-d6 (deuterated dimethyl sulfoxide) at 300 K, peptide concentration was 2-3 mg / ml, chemical shifts were measured relative to tetramethylsilane. Mass spectra were recorded on a VISION 2000 instrument (Termobioanalysis corp., Finnigan, USA) equipped with a class 3B pulsed nitrogen laser with a radiation wavelength of 337 nm, using the MALDI method using a 2,4,6-trihydroxyacetophenone matrix.

The synthesis of the peptide fragment of the chemokine TARC was carried out according to standard solid-phase synthesis technology using the Fmoc methodology. The Wang Fmoc aminoacyl polymer, a copolymer of styrene with 1% divinylbenzene with a hydroxymethyl phenoxymethyl anchor group, with a particle size of 200-400 mesh from Bachem (Switzerland), was used as a polymer carrier. The starting amino acid content was 0.7 mmol / g of aminoacylpolymer.

The synthesis was performed on a Tribute-UV automated peptide synthesizer from Protein Technologies (USA) according to the standard program for a single condensation of 0.5 mmol Fmoc amino acids. Used a 4-fold excess of Fmoc-protected amino acids in relation to the content of amino groups in the polymer. The synthesis was carried out from the C-terminus in accordance with the automatic synthesizer protocol for condensation by the TBTU / NMM method (2- (1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium tetrafluoroborate / N-methylmorpholine). For the release of α-amino groups during solid-phase synthesis (TFS), a solution of 10% 4-MePip and 2% DBU (1,8-diazabicyclo [5.4.0] undec-7-ene) in DMA (N, N-dimethylacetamide) was used.

Solid Phase Synthesis Protocol

At the end of TFS, the peptidyl polymer was transferred from the reactor to a Schott filter and washed with DMA (3 × 10 ml), DCM (3 × 10 ml), dried in air.

The final release and cleavage of the peptide from the polymer was carried out in one step by treating the corresponding peptidyl polymer with 10 ml of a mixture of 95% TFA, 2.5% deionized water, 2.5% TIBS for 1.5 h. Then the polymer was filtered off, washed with 2 × 2 ml a deblocking mixture, the filtrate was evaporated and dry diethyl ether was added to the residue. The precipitate was filtered off, washed with DCM (3 × 3 ml), ether (3 × 5 ml), dried in a vacuum desiccator. The crude product TFS contained, according to HPLC, about 80% of the basic substance. Purification of the obtained compound was carried out by preparative HPLC.

The structure of the obtained compound was confirmed by ¹ H-NMR spectroscopy and mass spectrometry under the above conditions. The yield of the synthesized TARC fragment 17-28, calculated on the starting amino acid attached to the polymer, was 46.2%. Mass spectrometry data: found m / z = 1446.16, calculated M = 1445.79. Analytical HPLC data: peptide retention time (R _t ) - 12.7 min, purity - 96.1%

Example 2

Testing the synthesized peptide was carried out to assess its effect on the migration of TEM lymphocyte leukemia cells of the CEM.NKR line stimulated by TARC. The cells were kindly provided by the head. lab. of immunology and cell biotechnology of the Republican Scientific Practical Center of Epidemiology and Microbiology of the Ministry of Health of the Republic of Belarus Goncharov A.E. Cells were cultured in RPMI-1640 medium supplemented with 10% cow fetal serum, 2 mM glutamine, 100 U / ml penicillin and streptomycin (all Invitrogen reagents). Cell expression of CCR4 receptors was confirmed by direct immunofluorescence (staining with anti-CCR4 antibodies labeled with phycoerythrin, eBioscience) and flow cytometry.

The cell migration response was evaluated in a Transwell system with a pore diameter of 8 μm for 24-well plates (Corning, NY). For the experiment, RPMI-1640 medium was used with the addition of 1% cow fetal serum. A 5 nM TARC chemokine solution (R&D Systems) in the medium or a medium without TARC (control) was added to the bottom of the Transwell system. The peptide was introduced into the lower part of the Transwell system to a final concentration of 100 μM. 100 μl of cell suspension (10 million / ml) was placed on top of the system. The plates were incubated in an atmosphere of 5% CO ₂ at 37 ° C for 18 hours. Cells that migrated to the lower part of the Transwell system were counted in the Goryaev chamber. The number of promigrated cells was expressed in relative units (O.E.), taking for 1 O.E. the level of spontaneous cell migration in control samples.

For intergroup comparisons, the Mann-Whitney U test was used. Data were expressed as median (25; 75 percentile). For data processing, the Statistica 7 software package was used. The differences were considered statistically significant at p <0.05.

In the model system used, TARC stimulated the migration of lymphoid cells. The number of cells in the lower part of Transwell was 7-8 times higher compared to the control. The obtained peptide fragment of the chemokine TARC suppressed TARC-induced migration by about 2 times, but did not affect the spontaneous cell motility. The indicated peptide did not affect cell migration stimulated by other chemokines, in particular, SDF-1 (data not shown).

In FIG. 1. The effect of the peptide H-Gly-Ala-Ile-Pro-Leu-Arg-Lys-Leu-Lys-Thr-Trp-Tyr-OH on the migration of CEM.NKR T-lymphoblastic leukemia cells stimulated by the TARC chemokine is shown. Experiment points are located along the X axis. At the top of the Transwell system, a suspension of cells in RPMI-1640 medium was added with the addition of 1% fetal bovine serum. In the lower part: the same environment (control) (point 1); TARC chemokine (5 nM) (2); TARC chemokine (5 nM) and peptide H-Gly-Ala-Ile-Pro-Leu-Arg-Lys-Leu-Lys-Thr-Trp-Tyr-OH (100 μM) (3); peptide H-Gly-Ala-Ile-Pro-Leu-Arg-Lys-Leu-Lys-Thr-Trp-Tyr-OH (100 μM) (4). The y axis shows the number of cells migrated, expressed in relative units (O.E.) 1 O.E. - the number of promigrated cells in the control samples.

BIBLIOGRAPHY

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Claims (2)

1. The peptide of the formula H-Gly-Ala-Ile-Pro-Leu-Arg-Lys-Leu-Lys-Thr-Trp-Tyr-OH, with the ability to inhibit cell migration stimulated by the chemokine TARC. 2. A method for producing a peptide according to claim 1 by the solid-phase method using Fmoc amino acids.

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