WO2021251850A1 - Specific peptide inhibitors of cysteine cathepsins - Google Patents
Specific peptide inhibitors of cysteine cathepsins Download PDFInfo
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- WO2021251850A1 WO2021251850A1 PCT/RU2021/050257 RU2021050257W WO2021251850A1 WO 2021251850 A1 WO2021251850 A1 WO 2021251850A1 RU 2021050257 W RU2021050257 W RU 2021050257W WO 2021251850 A1 WO2021251850 A1 WO 2021251850A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/005—Enzyme inhibitors
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/07—Tetrapeptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
- C07K5/101—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6472—Cysteine endopeptidases (3.4.22)
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/2201—Streptopain (3.4.22.10)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22015—Cathepsin L (3.4.22.15)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22016—Cathepsin H (3.4.22.16)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Definitions
- 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.
- Cysteine cathepsins are homologous proteinases of the CA clan of cysteine peptidases expressed in various cells and tissues of many species of organisms [Rawlings, ND, Barrett, AJ, Thomas, PD, Huang, X., Bateman, A., and Finn, RD (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.].
- cysteine cathepsins In the human genome, 11 cysteine cathepsins have been identified: B, C, F, I, K, L, O, S, V, W, and X (Z), which exhibit various catalytic activity.
- cathepsins F, O, S, K, V, L and W are endopeptidases with a wide substrate specificity; cathepsins I 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.
- cathepsins B, H, L, S, K are involved in the process of apoptosis [Voy, R., and Kroemer, G. (2008) Lysosomal membrane permeabilization in cell death, Oncogene, 27, 6434-6451; Repnik, U., Cesen, MH, and Turk, B. (2013) The Endolysosomal System in Cell Death and Survival, Cold Spring Harb. Perspect.
- 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.
- 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.
- 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.
- cysteine cathepsins are promising targets in the development of anticancer drugs aimed at inhibiting these enzymes.
- 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].
- 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 claimed invention can be used to prepare pharmaceutical compositions for inhibiting 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.
- peptides are 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.
- FIG. 1 shows the structures of specific peptide inhibitors of cysteine cathepsins Ac-PLVE-FMK and Ac-VLPE-FMK.
- Figure 2 shows graphs of the dependence of the fluorescence intensity of the hydrolysis product (free label of 7-amino-4-methylcumarine (AMK), cu) cysteine cathepsins (CtsB and CtsL) of the fluorogenic substrate Ac-PLVQ-AMK versus time (min), characterizing the inhibitory activity of 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 indices of the results of a "scratch" test on a monolayer of 769-P kidney tumor cells.
- Figure 4 shows an image and numerical indicators of the results of the "scratch" test on a monolayer of tumor cells of the kidney line A498.
- 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).
- FMK fluoromethyl ketone
- 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 and dp., Tetrahedron Letters 29, 1988, pp. 4005-4008; Mergler and dp., Tetrahedron Letters 29, 1988, pp. 4009-4012; Peptides, Chemistry and Biology, under ped. Kamber and dp., ESCOM, Leiden, 1992, pp.
- 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 the synthetic model peptide substrate of wheat cysteine proteinase triticaine-alpha Ac-PLVQ-AMK conjugated with 7-Amino-4-methylcoumarin (AMK), with the determination of hydrolysis products by the intensity of free AMK fluorescence described earlier [Patent RU 2603054.].
- red lines and detect the fluorescence intensity at room temperature at the excitation wavelength fluorescence of 360 nm and a fluorescence emission wavelength of 460 nm (the amount of hydrolyzed substrate Ac-PLVQ-AMK is determined by the fluorescence intensity; the reaction rate at the need is determined by the graph of the dependence of the amount of substrate (mol) on the time of hydrolysis (s) with subsequent processing of the data obtained using the linear regression method).
- the fluorescence intensity noticeably decreased in the presence of the claimed tetrapeptides (blue and green lines), which indicates an 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.
- Example 2 Evaluation of the inhibitory activity of Ac-PLVE-FMK and Ac-VLPE-FMK using tumor cell lines.
- cysteine cathepsins 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.
- the monolayer is washed twice with phosphate-buffered saline to remove detached cells and debris, and inhibitory tetrapeptides Ac-PLVE-FMK and Ac-VLPE-FMK are added at a final concentration of 20 ⁇ M, 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).
- 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.
Abstract
The invention relates to bioengineering. The use of synthetic peptides (peptide compounds) as specific inhibitors of cysteine cathepsins is proposed. The synthetic peptides are compounds of general formula R1-PEPT-R2, where PEPT is one of the amino acid sequences: PLVE and VLPE; R1 is one of the protecting groups: acetyl or benzyloxycarbonyl, or is absent; and R2 is derivatives of fluoromethyl ketone (FMK) or chloromethyl ketone (CMK). The proposed peptides have high affinity for the catalytic triad of cysteine cathepsins and can be used to make therapeutic agents which are specific inhibitors of proteolytic processes involving cysteine cathepsins, in particular oncogenic processes.
Description
СПЕЦИФИЧЕСКИЕ ПЕПТИДНЫЕ ИНГИБИТОРЫ ЦИСТЕИНОВЫХ SPECIFIC PEPTIDE CYSTEINE INHIBITORS
КАТЕПСИНОВ KATEPSINOV
Область техники, к которой относится изобретение The technical field to which the invention relates
Изобретение относится к биоинженерии, а именно к пептидам (пептидным соединениям), обладающим высокой аффинностью к каталитической триаде цистеиновых катепсинов, и может быть использовано для создания терапевтических средств, являющихся специфическими ингибиторами протеолитических процессов с участием цистеиновых катепсинов. 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.
Уровень техники Цистеиновые катепсины являются гомологичными протеиназами клана СА цистеиновых пептидаз, экспрессирующимися в различных клетках и тканях многих видов организмов [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.]. В геноме человека идентифицировано 11 цистеиновых катепсинов: В, С, F, И, К, L, О, S, V, W и X(Z), которые проявляют разнообразную каталитическую активность. Так, катепсины F, О, S, К, V, L и W представляют собой эндопептидазы с широкой субстратной специфичностью; катепсины И и В обладают как эндо-, так и экзопептидазной активностью, тогда как катепсины С и X являются исключительно амино- и карбоксипептидазой соответственно. Наибольшую активность цистеиновые катепсины проявляют в средах с пониженными значениями pH, вследствие чего изначально считалось, что функционирование этих ферментов ограничивается лизосомами и эндосомами. Однако появляется все больше данных о высокоспецифичной и направленной протеолитической активности катепсинов в других компартментах клеток, таких как секреторные везикулы, цитозоль и ядро, а также вне клеток [Spira, D., Stypmann, J., Tobin, D.J., Petermann, F, Mayer, C., Hagemann, S., Vasiljeva, O., Gunther, T., Schule, 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.]. Основной функцией катепсинов является низкоспецифичная деградация белков в лизосоме, однако некоторые представители этой группы ферментов выполняют и другие функции. Так, например, катепсины В, Н, L, S, К задействованы в процессе апоптоза [Воуа, Р., 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.], а катепсины S, F, L и V участвуют в презентации антигенов МНС класса II [Sadegh-Nasseri, S., and Kim, А. (2015) МНС class II auto-antigen presentation is unconventional, Front. Immunol., 6, 372.]. BACKGROUND Cysteine cathepsins are homologous proteinases of the CA clan of cysteine peptidases expressed in various cells and tissues of many species of organisms [Rawlings, ND, Barrett, AJ, Thomas, PD, Huang, X., Bateman, A., and Finn, RD (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, I, 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 I 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, F, Mayer , C., Hagemann, S., Vasiljeva, O., Gunther, T., Schule, 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 [Voy, R., and Kroemer, G. (2008) Lysosomal membrane permeabilization in cell death, Oncogene, 27, 6434-6451; Repnik, U., Cesen, MH, 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.].
Таким образом, цистеиновые катепсины выполняют различные функции в клетках и тканях, однако их активность должна строго регулироваться. Совокупность таких факторов как локализация (преимущественно в лизосомах), экспрессия эндогенных ингибиторов (цистатинов) низкие уровни pH (для оптимальной работы) катепсинов ограничивают область их действия и позволяют организму предотвращать деградацию белков цитоплазмы и межклеточного матрикса. Тем не менее любой сбой в регуляции, выраженной, например, в повышение уровня экспрессии и активности катепсинов часто связан с развитием различных патологических состояний, таких как неврологические расстройства, сердечно-сосудистые заболевания, ожирение, ревматоидный артрит и опухолевые заболевания. В частности, при онкогенезе цистеиновые катепсины детектируются в микроокружении опухолей, где они участвуют в процессах пролиферации, инвазии и метастазирования, обеспечивая деградацию внеклеточного матрикса, разрушение межклеточных взаимодействий и стимулируя ангиогенез. В связи с этим, цистеиновые катепсины являются перспективными мишенями при разработке противоопухолевых препаратов, направленных на ингибирование этих ферментов. 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.
Известны различные препараты, которые являются ингибиторами цистеиновых катепсинов. В основном это низкомолекулярные соединения, основанные на структурах эпоксисукцинила, винилсульфона или нитрила, действующие по принципу связывания с активным центром ферментов. Кроме того, для некоторых катепсинов были разработаны ингибиторы на основе антител. Однако все эти ингибиторы имеют различную специфичность, а также биологические свойства, связанные с проницаемостью в клетки и обладающие значительными побочными эффектами, ограничивающими возможности применения таких ингибиторов на практике. [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.]. 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 practical use of such inhibitors. [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.].
Кроме того, для ингибирования цистеиновых катепсинов (К, S, L) авторами патентов [RU 2692799, RU 2535479, RU 2399613] предложены конкретные соединения непептидного происхождения, которые предлагается использовать для
лечения или профилактики катепсин-зависимых заболеваний или состояний у млекопитающих. Разнообразие представленных модификаций этих соединений также обусловлено их различной специфичностью и биологическими свойствами при тех или иных условиях использования. Наиболее близкими к предлагаемому решению являются универсальный ингибитор каспаз, имеющий структуру Z-VAD-FMK ( -бензоилоксикарбонил-Уа! - Ala-Asp-трифторметилкетон) [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], который предотвращает апоптоз, а также пептидные ингибиторы каспаз: z- DEVD.fmk, Ac-YVAD.cmk. Каспазы относятся к семейству цистеиновых протеиназ, расщепляющих белки после аспартата. С помощью пептидных производных фторметилкетонов и хлорметилкетонов (напоминающими место расщепления известных каспазных субстратов), необратимо алкилирующих остаток цистеина в активном участке каспазы, было показано эффективное ингибирование этих ферментов. Ингибирующий эффект z-VAD.fmk, z-DEVD.fmk и Ac-YVAD.cmk также был показан и для катепсинов В и Н [Schotte Р. et al. Non-specific effects of methyl ketone peptide inhibitors of caspases //FEBS letters. - 1999. - T. 442. - JV°. 1. - C. 117- 121. Для бензилоксикарбонил-фенил-аланил-фторметилкетона (z-FA-FMK) помимо подавления процессов апоптоза и воспаления посредством ингибирования некоторых каспаз, также показана способность ингибировать катепсин В [The Cathepsin В 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]. Однако в связи с тем, что вышеуказанные пептидные ингибиторы были созданы для ингибирования членов семейства каспаз, их специфичности может быть не вполне достаточно для эффективного ингибирования ферментов семейства цистеиновых катепсинов. In addition, to inhibit 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 treating or preventing 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 (-benzoyloxycarbonyl-Ya! - 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 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. - JV °. 1. - C. 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.
Технической проблемой, на решение которой направлено предлагаемое изобретение, является создание новых пептидных последовательностей, являющихся специфическими ингибиторами цистеиновых катепсинов, т.е. способных ингибировать функциональную (протеолитическую) активность цистеиновых катепсинов, с перспективой использования в разработке противоопухолевых терапевтических средств.
Раскрытие сущности изобретения 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
Техническим результатом изобретения является создание специфических пептидов (пептидных соединений), обладающих ингибирующим действием на функциональную активность цистеиновых катепсинов. 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.
Технический результат достигается за счет синтеза специфических пептидных соединений - производных фторметилкетонов или хлорметилкетонов тетрапептидов структуры R1-PEPT-R2, где РЕРТ - одна из аминокислотных последовательностей: PLVE, VLPE; R1 - одна из защитных групп: ацетильная (Ас), бензилоксикарбонильная (Z) или отсутствует; R2 - производные фторметилкетона (FMK) или хлорметилкетона (СМК), характеризующихся способностью ингибировать протеолитическую активность цистеиновых катепсинов. 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 (SMK), characterized by the ability to inhibit the proteolytic activity of cysteine cathepsins.
Заявленное изобретение может применяться для приготовления фармацевтических композиций для подавления специфической активности цистеиновых катепсинов, содержащих пептидное соединение и фармацевтически приемлемый носитель. Пептидные соединения могут применяться для приготовления лекарственных средств, вызывающих подавление специфической активности цистеиновых катепсинов, в терапии опухолевых заболеваний. The claimed invention can be used to prepare pharmaceutical compositions for inhibiting 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.
Получение таких модифицированных пептидов легко осуществимо стандартными методами пептидного синтеза. Пептидная природа специфических ингибиторов позволяет использовать их в качестве действующего вещества для разработок фармкомпозиций. Преимуществами пептидов перед другими типами препаратов состоит в том, что они, в основном, безопасны, быстро выводятся из организма и имеют значительно меньше побочных эффектов, чем средства непептидной природы. Большинство пептидных лекарств вводят парентеральным путем, но с развитием соответствующих технологий разрабатываются и иные формы введения: пероральный, интраназальный, трансдермальный, т.е. возможен подбор вариантов места действия пептида конкретно под определенные условия применения. 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.
Краткое описание чертежей Brief Description of Drawings
Изобретение поясняется иллюстрациями, где на Фиг. 1 представлены структуры специфических пептидных ингибиторов цистеиновых катепсинов Ас- PLVE-FMK и Ac-VLPE-FMK. 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.
На Фиг.2 представлены графики зависимости интенсивности флуоресценции продукта гидролиза (свободной метки 7-Амино-4-метилку марина (АМК), у.е.)
цистеиновыми катепсинами (CtsB и CtsL) флуорогенного субстрата Ac-PLVQ-AMK от времени (мин), характеризующие ингибирующую активность ферментов, где а) график зависимости для катепсина В (CtsB), б) график зависимости для катепсина L (CtsL), с) график зависимости в отсутствии ферментов (в качестве контроля отсутствия флуоресценции метки); красными линиями показана активность ферментов без добавления ингибиторов, синими - в присутствии пептидного ингибитора структуры Ac-PLVE-FMK, зелеными - в присутствии пептидного ингибитора структуры Ac-VLPE-FMK. Figure 2 shows graphs of the dependence of the fluorescence intensity of the hydrolysis product (free label of 7-amino-4-methylcumarine (AMK), cu) cysteine cathepsins (CtsB and CtsL) of the fluorogenic substrate Ac-PLVQ-AMK versus time (min), characterizing the inhibitory activity of 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.
На Фиг.З представлены изображение и численные показатели результатов «скретч»-теста на монослое опухолевых клеток почки линии 769-Р. Fig. 3 shows an image and numerical indices of the results of a "scratch" test on a monolayer of 769-P kidney tumor cells.
На Фиг.4 представлены изображение и численные показатели результатов «скретч»-теста на монослое опухолевых клеток почки линии А498. Figure 4 shows an image and numerical indicators of the results of the "scratch" test on a monolayer of tumor cells of the kidney line A498.
Осуществление изобретения Implementation of the invention
Изобретение иллюстрируется с использованием соединений R1-PEPT-R2, где РЕРТ - одна из аминокислотных последовательностей: PLVE, VLPE; R1 -ацетильная группа (Ac); R2 - производные фторметилкетона (FMK), т.е. Ac-PLVE-FMK (Acetyl - Pro-Leu- Val-Glu-FMK) и Ac-VLPE-FMK (Acetyl-Val-Leu-Pro-Glu-FMK). 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).
На примере изучения функциональной активности цистеиновой протеиназы пшеницы Тритикаина-альфа были определены аминокислотные последовательности с предпочтительными сайтами расщепления субстратов [Savvateeva LV, Gorokhovets NV, Makarov VA, Serebryakova MV, Solovyev AG, Morozov SY, Reddy VP, Zernii EY, Zamyatnin AA Jr, Aliev G. (2015) Glutenase and collagenase activities of wheat cysteine protease Triticain-a: feasibility for enzymatic therapy assays. Int J Biochem Cell Biol., 62:115-24.]. На основе полученных данных посредством компьютерного моделирования (с помощью программы PLANTS [Korb, О.; Stutzle, Т.; Exner, Т.Е. Empirical scoring functions for advanced protein- ligand docking with PLANTS. Journal of chemical information and modeling 2009, 49, 84-96.] для моделирования белково- лигандного взаимодействия) были подобраны пептидные последовательности, обладающие наибольшей аффинностью к каталитическому центру цистеиновых протеиназ, в частности тетрапептиды PLVQ(E) и VLPQ(E). Путем конъюгирования фторметилкетоновой группы (FMK) к данным пептидам PLVE и VLPE были получены новые высокоспецифические ингибиторы цистеиновых протеиназ (Фиг.1). Механизм необратимого ингибирования осуществляется за счет ковалентного связывания каталитического цистеина с FMK [Rasnick, D. Synthesis of peptide
fluoromethyl ketones and the inhibition of human cathepsin B. Analytical biochemistry 1985, 149, 461-465.]. 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-a: 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, T.E. 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 (Figure 1). The mechanism of irreversible inhibition is carried out 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.].
Пептиды получали стандартными методами химического синтеза (твердофазным или в жидкой фазе), при котором пептиды получают путем соединения различных аминокислот друг с другом с последующей химической модификацией для введения функциональных групп. Способы химического синтеза пептидов широко известны и хорошо описаны с помощью известных в данной области техники методов [US 6015881; Mergler и dp., Tetrahedron Letters 29, 1988, сс.4005-4008; Mergler и dp., Tetrahedron Letters 29, 1988, сс.4009-4012; Peptides, Chemistry and Biology, под ped. Kamber и dp., изд-во ESCOM, Leiden, 1992, cc.525- 526; Riniker и dp., Tetrahedron Letters 49, 1993, cc.9307-9320; Lloyd-Williams и dp., Tetrahedron Letters 49, 1993, cc.11065-11133, и Andersson и dp., Biopolymers 55, 2000, cc.227-250]. 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 and dp., Tetrahedron Letters 29, 1988, pp. 4005-4008; Mergler and dp., Tetrahedron Letters 29, 1988, pp. 4009-4012; Peptides, Chemistry and Biology, under ped. Kamber and dp., ESCOM, Leiden, 1992, pp. 525-526; Riniker and dp., Tetrahedron Letters 49, 1993, 9307-9320; Lloyd-Williams and dp., Tetrahedron Letters 49, 1993, 11065-11133, and Andersson and dp., Biopolymers 55, 2000, 227-250].
Ингибирующие свойства Ас-PL VE-FMK и Ac-VLPE-FMK оценивали in vitro биохимическими методами и на опухолевых линиях клеток. В качестве модельных ферментов были взяты рекомбинантные цистеиновые катепсины L и В человека (CtsL и CtsB), так как они проявляют как эндо-, так и эндо -/экзопептидазную активность соответственно. The inhibitory properties of Ac-PL VE-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.
Пример 1. Определение ингибирующей активности Ac-PLVE-FMK и Ас- VLPE-FMK с использованием специфического флуорогенного субстрата. Example 1. Determination of the inhibitory activity of Ac-PLVE-FMK and Ac-VLPE-FMK using a specific fluorogenic substrate.
Активность рекомбинантных катепсинов Cts В и L определяли по способности гидролизовать синтетический модельный пептидный субстрат цистеиновой протеиназы пшеницы тритикаина-альфа Ac-PLVQ-AMK, конъюгированный с 7-Амино-4-метилкумарином (АМК), с определением продуктов гидролиза по интенсивности флуоресценции свободного АМК как описано ранее [Патент RU 2603054.]. Так, к 20 нМ раствору рекомбинантного Cts В или Cts L в 0,1М натрий-ацетатного буфере с содержанием 100 мМ NaCl, 0,5% DMSO, 0,6мМ ЭДТА, pH 4,6 в присутствии и отсутствии 2 мкМ тетрапептидных ингибиторов Ac- PLVE-FMK (Фиг.2, синие линии) и Ac-VLPE-FMK (Фиг.2, зеленые линии) добавляют 50 mM флуорогенного субстрата (Фиг.2, красные линии) и детектируют интенсивность флуоресценции при комнатной температуре при длине волны возбуждения флуоресценции, равной 360 нм, и длине волны испускания флуоресценции, равной 460 нм (количество гидролизованного субстрата Ac-PLVQ- AMK определяют по интенсивности флуоресценции; скорость реакции при
необходимости определяют по графику зависимости количества субстрата (моль) от времени гидролиза (с) с последующей обработкой полученных данных с применением метода линейной регрессии). The activity of the recombinant cathepsins Cts B and L was determined by the ability to hydrolyze the synthetic model peptide substrate of wheat cysteine proteinase triticaine-alpha Ac-PLVQ-AMK conjugated with 7-Amino-4-methylcoumarin (AMK), with the determination of hydrolysis products by the intensity of free AMK fluorescence 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 mM fluorogenic substrate (Fig. 2, red lines) and detect the fluorescence intensity at room temperature at the excitation wavelength fluorescence of 360 nm and a fluorescence emission wavelength of 460 nm (the amount of hydrolyzed substrate Ac-PLVQ-AMK is determined by the fluorescence intensity; the reaction rate at the need is determined by the graph of the dependence of the amount of substrate (mol) on the time of hydrolysis (s) with subsequent processing of the data obtained using the linear regression method).
Как видно из рисунка Фиг. 2 и таблицы 1, интенсивность флуоресценции заметно снижалась в присутствии заявляемых тетрапептидов (синие и зеленые линии), что говорит об активной конкуренции субстрата и ингибиторов за активный центр фермента, и, следовательно, свидетельствует о специфичном и эффективном подавлении протеиназной активности цистеиновых катепсинов. As seen in FIG. 2 and Table 1, the fluorescence intensity noticeably decreased in the presence of the claimed tetrapeptides (blue and green lines), which indicates an 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.
Таблица 1
Table 1
Пример 2. Оценка ингибирующей активности Ac-PLVE-FMK и Ac-VLPE- FMK с использованием опухолевых клеточных линий. Example 2. Evaluation of the inhibitory activity of Ac-PLVE-FMK and Ac-VLPE-FMK using tumor cell lines.
Известно, что эффективное ингибирование активности цистеиновых катепсинов ассоциировано с изменениями определенных свойств опухолевых клеток, в частности, инвазии и подвижности. 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.
Для исследования влияния Ac-PLVE-FMK и Ac-VLPE-FMK на подвижность клеток использовали scratch-тест («скретч-тест»): на монослой клеток рака почки линий 769-Р и А498, выращенных в ростовой среде RPMI 1640 с добавлением 10% бычьей сыворотки и 1% содержанием антибиотиков пенициллин-стрептомицин в инкубаторе с 5% С02 при 37°С, наносят царапину с помощью наконечника пипетки. Монослой промывают дважды фосфатно-солевым буфером для удаления открепившихся клеток и дебриса и добавляют ингибиторные тетрапептиды Ac- PLVE-FMK и Ac-VLPE-FMK в конечной концентрации 20 мкМ, после чего клетки продолжают наращивать в С02-инкубаторе, детектируя «заживление» клеточного
монослоя с использованием микроскопа в определенные временные интервалы (Фиг.З, 4, таблица 2, 3). 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% penicillin-streptomycin antibiotics in an incubator with 5% CO2 at 37 ° C, scratch with a pipette tip. The monolayer is washed twice with phosphate-buffered saline to remove detached cells and debris, and 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).
Таблица 2
Таблица 3
table 2 Table 3
Как видно из Фиг. 3, 4 и таблиц 2, 3, добавление тетрапептидов замедляло рост образования монослоя опухолевых клеток, т.е. пептидные ингибиторы эффективно препятствовали «заживлению» (закрытию зазора) как через 8, так и через 24 часа после образования царапины. Для наилучшей репрезентативности данных были построены гистограммы, отражающие процентное соотношение зарегистрированных изменений в размерах повреждения клеточного монослоя по сравнению с контролем (клетки без обработки тетрапептидами). Таким образом, можно утверждать, что ингибирование Cts с использованием тетрапептидов в опухолевых клетках может влиять на адгезию клеток, препятствуя образованию межклеточных контактов. 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 scratch was formed. For the best representativeness of the data, histograms were constructed, reflecting the percentage ratio 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.
Таким образом, заявленные специфические пептидные ингибиторы, обладающие способностью ингибировать цистеиновые катепсины, могут служить основой фармацевтических композиций для разработки и получения лекарственных средств для лечения заболеваний, связанных с повышенной активностью цистеиновых катепсинов, в частности, противоопухолевых препаратов.
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
1. Пептидное соединение с общей формулой R1-PEPT-R2, где РЕРТ - одна из аминокислотных последовательностей: PLVE, VLPE; R1 - одна из защитных групп: ацетильная, бензилоксикарбонильная или отсутствует; R2 - производные фторметилкетона (FMK) или хлорметилкетона (СМК), характеризующееся способностью ингибировать протеолитическую активность цистеиновых катепсинов. 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 (SMK), characterized by the ability to inhibit the proteolytic activity of cysteine cathepsins.
2. Применение пептидного соединения по п. 1 в качестве ингибитора протеолитической активности цистеиновых катепсинов. 2. The use of a peptide compound according to claim 1 as an inhibitor of the proteolytic activity of cysteine cathepsins.
3. Применение пептидного соединения по п. 1 для терапии опухолевых заболеваний. 3. The use of a peptide compound according to claim 1 for the therapy of tumor diseases.
4. Применение пептидного соединения по п. 1 для приготовления лекарственного средства, вызывающего подавление специфической активности цистеиновых катепсинов. 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.
5. Фармацевтическая композиция для подавления специфической активности цистеиновых катепсинов, содержащая пептидное соединение по п.1, и фармацевтически приемлемый носитель.
5. A pharmaceutical composition for suppressing the specific activity of cysteine cathepsins, comprising a peptide compound according to claim 1 and a pharmaceutically acceptable carrier.
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