RU2752531C1 - Specific peptide inhibitors of cysteine cathepsins - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology. A peptide compound as a specific inhibitor of cysteine cathepsins and its applications are proposed. Synthetic peptides are R1-PEPT-R2 compounds, where PEPT is one of the amino acid sequences: PLVE, VLPE; R1 is one of the protective groups: acetyl, benzyloxycarbonyl or none; R2 is a derivative of fluoromethyl ketone (FMK) or chloromethyl ketone (CMK). The proposed peptides are able to inhibit the proteolytic activity of cysteine cathepsins, are characterized by high affinity to the catalytic triad of cysteine cathepsins, and can be used to create drugs that are specific inhibitors of proteolytic processes involving cysteine cathepsins, in particular, oncogenesis processes.

EFFECT: creation of drugs that are specific inhibitors of proteolytic processes involving cysteine cathepsins.

4 cl, 4 dwg, 3 tbl, 2 ex

Description

The technical field to which the invention relates

The invention relates to bioengineering, namely to peptides (peptide compounds) with high affinity for the catalytic triad of cysteine cathepsins, and can be used to create therapeutic agents that are specific inhibitors of proteolytic processes involving cysteine cathepsins.

State of the art

Cysteine cathepsins are homologous proteinases of the CA clan of cysteine peptidases expressed in various cells and tissues of many species of organisms [Rawlings, N.D., Barrett, A.J., Thomas, P.D., Huang, X., Bateman, A., and Finn, R.D. (2018) The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database, Nucleic Acids Res., 46, D624-D632.]. In the human genome, 11 cysteine cathepsins have been identified: B, C, F, H, K, L, O, S, V, W, and X (Z), which exhibit various catalytic activity. Thus, cathepsins F, O, S, K, V, L and W are endopeptidases with a wide substrate specificity; cathepsins H and B have both endo- and exopeptidase activity, while cathepsins C and X are exclusively amino and carboxypeptidase, respectively. Cysteine cathepsins are most active in environments with low pH values, as a result of which it was initially believed that the functioning of these enzymes is limited to lysosomes and endosomes. However, there is more and more data on highly specific and targeted proteolytic activity of cathepsins in other cell compartments, such as secretory vesicles, cytosol and nucleus, as well as outside cells [Spira, D., Stypmann, J., Tobin, DJ, Petermann, I., Mayer, C., Hagemann, S., Vasiljeva, O., Günther, T., Schüle, R., Peters, C., and Reinheckel, T. (2007) Cell type-specific functions of the lysosomal protease cathepsin L in the heart, J. Biol. Chem., 282, 37045-37052.]. The main function of cathepsins is low-specific degradation of proteins in the lysosome; however, some members of this group of enzymes also perform other functions. For example, cathepsins B, H, L, S, K are involved in the process of apoptosis [Boya, P., and Kroemer, G. (2008) Lysosomal membrane permeabilization in cell death, Oncogene, 27, 6434-6451; Repnik, U., Cesen, M.H., and Turk, B. (2013) The Endolysosomal System in Cell Death and Survival, Cold Spring Harb. Perspect. Biol., 5, a008755-a008755.], And cathepsins S, F, L and V are involved in the presentation of MHC class II antigens [Sadegh-Nasseri, S., and Kim, A. (2015) MHC class II auto-antigen presentation is unconventional, Front. Immunol., 6, 372.].

Thus, cysteine cathepsins perform various functions in cells and tissues, but their activity must be strictly regulated. A combination of factors such as localization (mainly in lysosomes), expression of endogenous inhibitors (cystatins), low pH levels (for optimal performance) of cathepsins limit their area of action and allow the body to prevent the degradation of cytoplasmic proteins and extracellular matrix. Nevertheless, any malfunction in regulation, expressed, for example, in an increase in the level of expression and activity of cathepsins, is often associated with the development of various pathological conditions, such as neurological disorders, cardiovascular diseases, obesity, rheumatoid arthritis, and tumor diseases. In particular, during oncogenesis, cysteine cathepsins are detected in the microenvironment of tumors, where they participate in the processes of proliferation, invasion and metastasis, providing degradation of the extracellular matrix, destruction of intercellular interactions and stimulating angiogenesis. In this regard, cysteine cathepsins are promising targets in the development of anticancer drugs aimed at inhibiting these enzymes.

Various drugs are known that are inhibitors of cysteine cathepsins. Basically, these are low molecular weight compounds based on the structures of epoxysuccinyl, vinyl sulfone or nitrile, acting on the principle of binding to the active center of enzymes. In addition, antibody-based inhibitors have been developed for some cathepsins. However, all these inhibitors have different specificities, as well as biological properties associated with permeability into cells and have significant side effects that limit the possibilities of using such inhibitors in practice. [Petushkova AI, Savvateeva LV, Korolev DO, Zamyatnin AA Jr. (2019) Cysteine Cathepsins: Potential Applications in Diagnostics and Therapy of Malignant Tumors. Biochemistry (Mosc) .; 84 (7): 746-761.].

In addition, for the inhibition of cysteine cathepsins (K, S, L), the authors of patents [RU 2692799, RU 2535479, RU 2399613] proposed specific compounds of non-peptide origin, which are proposed to be used for the treatment or prevention of cathepsin-dependent diseases or conditions in mammals. The variety of the presented modifications of these compounds is also due to their different specificity and biological properties under certain conditions of use.

Closest to the proposed solution are a universal caspase inhibitor having the structure Z-VAD-FMK (N-benzoyloxycarbonyl-Va1-Ala-Asp-trifluoromethyl ketone) [Van Noorden CJ. (2001) The history of Z-VAD-FMK, a tool for understanding the significance of caspase inhibition. Acta Histochem. 103 (3): 241-51], which prevents apoptosis, as well as peptide caspase inhibitors: z-DEVD.fmk, Ac-YVAD.cmk. Caspases belong to the family of cysteine proteinases that break down proteins after aspartate. Using peptide derivatives of fluoromethyl ketones and chloromethyl ketones (reminiscent of the cleavage site of known caspase substrates), irreversibly alkylating the cysteine residue in the active site of the caspase, effective inhibition of these enzymes was shown. The inhibitory effect of z-VAD.fmk, z-DEVD.fmk and Ac-YVAD.cmk has also been shown for cathepsins B and H [Schotte P. et al. Non-specific effects of methyl ketone peptide inhibitors of caspases // FEBS letters. - 1999. - T. 442. - No. 1. - S. 117-121. For benzyloxycarbonyl-phenyl-alanyl-fluoromethyl ketone (z-FA-FMK), in addition to suppressing the processes of apoptosis and inflammation by inhibiting some caspases, the ability to inhibit cathepsin B has also been shown [The Cathepsin B Inhibitor, z-FA-FMK, Inhibits Human T Cell Proliferation In Vitro and Modulates Host Response to Pneumococcal Infection In Vivo Clare P. Lawrence, Aras Kadioglu, Ai-Li Yang, William R. Coward, Sek C. Chow The Journal of Immunology September 15, 2006, 177 (6) 3827-3836].

However, due to the fact that the aforementioned peptide inhibitors were designed to inhibit members of the caspase family, their specificity may not be sufficient to effectively inhibit the enzymes of the cysteine cathepsin family.

The technical problem to be solved by the present invention is the creation of new peptide sequences that are specific inhibitors of cysteine cathepsins, i.e. capable of inhibiting the functional (proteolytic) activity of cysteine cathepsins, with the prospect of being used in the development of antitumor therapeutic agents.

Disclosure of the essence of the invention

The technical result of the invention is the creation of specific peptides (peptide compounds) that have an inhibitory effect on the functional activity of cysteine cathepsins.

The technical result is achieved through the synthesis of specific peptide compounds - derivatives of fluoromethyl ketones or chloromethyl ketones tetrapeptides of the structure R1-pept-R2, where PEPT is one of the amino acid sequences: PLVE, VLPE; R1 is one of the protective groups: acetyl (Ac), benzyloxycarbonyl (Z) or absent; R2 - derivatives of fluoromethyl ketone (FMK) or chloromethyl ketone (CMK), characterized by the ability to inhibit the proteolytic activity of cysteine cathepsins.

The claimed invention can be used for the preparation of pharmaceutical compositions for suppressing the specific activity of cysteine cathepsins containing a peptide compound and a pharmaceutically acceptable carrier. Peptide compounds can be used for the preparation of drugs that suppress the specific activity of cysteine cathepsins in the therapy of tumor diseases.

The preparation of such modified peptides is readily accomplished by standard peptide synthesis methods. The peptide nature of specific inhibitors allows them to be used as an active substance for the development of pharmaceutical compositions. The advantages of peptides over other types of drugs are that they are generally safe, quickly excreted from the body and have significantly fewer side effects than non-peptide drugs. Most peptide drugs are administered parenterally, but with the development of appropriate technologies, other forms of administration are being developed: oral, intranasal, transdermal, i.e. it is possible to select options for the site of action of the peptide specifically for certain conditions of use.

Brief Description of Drawings

The invention is illustrated by illustrations, where FIG. 1 shows the structures of specific peptide inhibitors of cysteine cathepsins Ac-PLVE-FMK and Ac-VLPE-FMK.

FIG. 2 shows the graphs of the dependence of the fluorescence intensity of the hydrolysis product (free label of 7-amino-4-methylcoumarin (AMC), c.u.) by cysteine cathepsins (CtsB and CtsL) of the fluorogenic substrate Ac-PLVQ-AMK on time (min), characterizing the inhibitory activity enzymes, where a) a graph of dependence for cathepsin B (CtsB), b) a graph of dependence for cathepsin L (CtsL), c) a graph of dependence in the absence of enzymes (as a control for the absence of fluorescence of the label); red lines show the activity of enzymes without the addition of inhibitors, blue - in the presence of a peptide inhibitor of the Ac-PLVE-FMK structure, green - in the presence of a peptide inhibitor of the Ac-VLPE-FMK structure.

FIG. 3 shows an image and numerical indicators of the results of a "scratch" test on a monolayer of 769-P kidney tumor cells.

FIG. 4 shows an image and numerical indicators of the results of the "scratch" test on a monolayer of kidney tumor cells of the A498 line.

Implementation of the invention

The invention is illustrated using compounds R1-pept-R2, where PEPT is one of the amino acid sequences: PLVE, VLPE; R1 is an acetyl group (Ac); R2 are fluoromethyl ketone (FMK) derivatives, i.e. Ac-PLVE-FMK (Acetyl-Pro-Leu-Val-Glu-FMK) and Ac-VLPE-FMK (Acetyl-Val-Leu-Pro-Glu-FMK).

Using the example of studying the functional activity of wheat cysteine proteinase Triticain-alpha, amino acid sequences with preferred cleavage sites of substrates were determined [Savvateeva LV, Gorokhovets NV, Makarov VA, Serebryakova MV, Solovyev AG, Morozov SY, Reddy VP, Zernii EY, Zamyatnin AA Jr, Zernii EY, Zamyatnin AA Jr, G. (2015) Glutenase and collagenase activities of wheat cysteine protease Triticain-α: feasibility for enzymatic therapy assays. Int J Biochem Cell Biol. 62: 115-24.]. Based on the data obtained by means of computer modeling (using the PLANTS program [Korb, O .; Stutzle, T .; Exner, TE Empirical scoring functions for advanced protein- ligand docking with PLANTS. Journal of chemical information and modeling 2009, 49, 84- 96.] to model the protein-ligand interaction), peptide sequences with the highest affinity for the catalytic center of cysteine proteinases, in particular the tetrapeptides PLVQ (E) and VLPQ (E), were selected. New highly specific inhibitors of cysteine proteinases were obtained by conjugating a fluoromethyl ketone group (FMK) to these PLVE and VLPE peptides (Fig. 1). The mechanism of irreversible inhibition is due to the covalent binding of catalytic cysteine to FMK [Rasnick, D. Synthesis of peptide fluoromethyl ketones and the inhibition of human cathepsin B. Analytical biochemistry 1985, 149, 461-465.].

Peptides were prepared by standard chemical synthesis methods (solid phase or liquid phase), in which peptides are prepared by combining different amino acids with each other, followed by chemical modification to introduce functional groups. Methods for the chemical synthesis of peptides are widely known and well described using methods known in the art [US 6015881; Mergler et al., Tetrahedron Letters 29, 1988, cc. 4005-4008; Mergler et al., Tetrahedron Letters 29, 1988, cc. 4009-4012; Peptides, Chemistry and Biology, ed. Kamber et al., ESCOM, Leiden, 1992, cc. 525-526; Riniker et al., Tetrahedron Letters 49, 1993, cc. 9307-9320; Lloyd-Williams et al., Tetrahedron Letters 49, 1993, cc. 11065-11133, and Andersson et al., Biopolymers 55, 2000, cc. 227-250].

The inhibitory properties of Ac-PLVE-FMK and Ac-VLPE-FMK were assessed in vitro by biochemical methods and on tumor cell lines. Recombinant human cysteine cathepsins L and B (CtsL and CtsB) were taken as model enzymes, since they exhibit both endo- and endo- / exopeptidase activity, respectively.

Example 1. Determination of the inhibitory activity of Ac-PLVE-FMK and Ac-VLPE-FMK using a specific fluorogenic substrate.

The activity of the recombinant cathepsins Cts B and L was determined by the ability to hydrolyze a synthetic model peptide substrate of wheat cysteine proteinase triticaine-alpha Ac-PLVQ-AMK conjugated with 7-Amino-4-methylcoumarin (AMC), with the determination of hydrolysis products by the intensity of AMK fluorescence as described earlier [Patent RU 2603054.]. Thus, to a 20 nM solution of recombinant Cts B or Cts L in 0.1 M sodium acetate buffer containing 100 mM NaCl, 0.5% DMSO, 0.6 mM EDTA, pH 4.6 in the presence and absence of 2 μM tetrapeptide inhibitors Ac-PLVE-FMK (Fig. 2, blue lines) and Ac-VLPE-FMK (Fig. 2, green lines) add 50 μM fluorogenic substrate (Fig. 2, red lines) and detect the fluorescence intensity at room temperature at wavelength excitation of fluorescence equal to 360 nm and a wavelength of emission of fluorescence equal to 460 nm (the amount of hydrolyzed substrate Ac-PLVQ-AMK is determined by the fluorescence intensity; the reaction rate, if necessary, is determined from the graph of the dependence of the amount of substrate (mol) on the hydrolysis time (s) s subsequent processing of the obtained data using the linear regression method).

As seen in FIG. 2 and Table 1, the fluorescence intensity markedly decreased in the presence of the claimed tetrapeptides (blue and green lines), which indicates active competition between the substrate and inhibitors for the active center of the enzyme, and, therefore, indicates a specific and effective suppression of the proteinase activity of cysteine cathepsins.

Table 1

Time, min Fluorescence intensity, c.u. Blank (Substrate Ac-PLVQ-AMK (Sub) without enzyme) CtsB CtsL + Sub + Sub + Ac-PLVE-FMK + Sub + Ac-VLPE-FMK + Sub + Sub + Ac-PLVE-FMK + Sub + Ac-VLPE-FMK 0.3 0 3500 3000 3100 5000 4000 4300 3 0.1 4300 3700 3700 7300 6300 6400 6 0 5600 4700 4600 9500 7200 7800 nine 0.1 6300 4900 4800 11300 8200 9100 12 0 7000 5050 4950 14000 9200 9500

Example 2. Evaluation of the inhibitory activity of Ac-PLVE-FMK and Ac-VLPE-FMK using tumor cell lines.

It is known that effective inhibition of the activity of cysteine cathepsins is associated with changes in certain properties of tumor cells, in particular, invasion and motility.

To study the effect of Ac-PLVE-FMK and Ac-VLPE-FMK on cell motility, a scratch test was used: on a monolayer of renal cancer cells of lines 769-P and A498 grown in RPMI 1640 growth medium supplemented with 10 % bovine serum and 1% antibiotic penicillin-streptomycin in a 5% CO2 incubator at 37 ° C, scratch with a pipette tip. The monolayer is washed twice with phosphate-buffered saline to remove detached cells and debris, and the inhibitory tetrapeptides Ac-PLVE-FMK and Ac-VLPE-FMK are added at a final concentration of 20 μM, after which the cells continue to grow in a CO2 incubator, detecting the "healing" of the cell monolayer using a microscope at certain time intervals (Fig. 3, 4, table 2, 3).

table 2

Cells line 769-P Size of cell damage (scratches), microns Time, h Ctrl (without adding specific peptide inhibitors) Ac-PLVE-FMK Ac-VLPE-FMK 0 390 389 387 eight 236 300 277 24 fifteen 56 80

Table 3

Cell line A489 Size of cell damage (scratches), microns Time, h Ctrl (without adding specific peptide inhibitors) Ac-PLVE-FMK Ac-VLPE-FMK 0 508 514 520 eight 437 467 476 24 282 407 376

As seen in FIG. 3, 4 and tables 2, 3, the addition of tetrapeptides slowed down the growth of the formation of a monolayer of tumor cells, i.e. peptide inhibitors effectively prevented "healing" (gap closure) both 8 and 24 hours after the formation of the scratch. For the best representativeness of the data, histograms were constructed, reflecting the percentage of the registered changes in the size of damage to the cell monolayer in comparison with the control (cells without tetrapeptide treatment). Thus, it can be argued that inhibition of Cts using tetrapeptides in tumor cells can affect cell adhesion, preventing the formation of intercellular contacts.

Thus, the claimed specific peptide inhibitors capable of inhibiting cysteine cathepsins can serve as the basis for pharmaceutical compositions for the development and preparation of drugs for the treatment of diseases associated with increased activity of cysteine cathepsins, in particular, antitumor drugs.

Claims (4)

1. Peptide compound with the general formula R1-PEPT-R2, where PEPT is one of the amino acid sequences: PLVE, VLPE; R1 is one of the protective groups: acetyl, benzyloxycarbonyl or absent; R2 - derivatives of fluoromethyl ketone (FMK) or chloromethyl ketone (CMK), characterized by the ability to inhibit the proteolytic activity of cysteine cathepsins. 2. The use of a peptide compound according to claim 1 as an inhibitor of the proteolytic activity of cysteine cathepsins. 3. The use of a peptide compound according to claim 1 for the treatment of diseases associated with increased activity of cysteine cathepsins. 4. The use of a peptide compound according to claim 1 for the preparation of a medicament causing suppression of the specific activity of cysteine cathepsins.

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