RU2796104C1 - Application of peptide compound for apoptosis induction in a tumor cell - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to the use of peptide compound containing amino acid sequence FFSFGGAL to suppress activity of cathepsin S, which causes apoptosis induction in a tumor cell. Invention also relates to the use of peptide compound containing amino acid sequence FFSFGGAL for the preparation of a pharmaceutical composition for inhibition of cathepsin S activity, which causes apoptosis induction in a tumor cell.

EFFECT: therapeutic agent for treatment of diseases associated with impaired expression of cathepsin S and/or BCL-2 family proteins.

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Description

The field of technology to which the invention belongs

The invention relates to the pharmaceutical industry and can be used as a therapeutic agent for the treatment of diseases associated with impaired expression of cathepsin S and/or proteins of the BCL-2 family.

State of the art

Three aspects distinguish human tumors of various types and etiologies from normal cells: the ability to proliferate more rapidly, evasion of cell growth suppression, and increased sensitivity to cytotoxic therapy [Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. cell. 2011 Mar 4; 144(5): 646-74. doi: 10.1016/j.cell.2011.02.013. PMID: 21376230]. In addition, two very important processes give tumor cells a survival advantage. Firstly, cells exhibit chemotherapeutic resistance to a programmed cell death process called apoptosis, and secondly, such cells develop a unique property of increased motility, which contributes to their spread during metastasis [Norouzi S, Gorgi Valokala M, Mosaffa F, Zirak MR, Zamani P, Behravan J. Crosstalk in cancer resistance and metastasis. Crit Rev Oncol Hematol. Dec 2018; 132:145-153. doi: 10.1016/j.critrevonc.2018.09.017. Epub 2018 Oct 4. PMID: 30447920; Steeg PS. Targeting metastasis. Nat Rev Cancer. 2016 Apr; 16(4):201-18. doi: 10.1038/nrc.2016.25. PMID: 27009393; PMCID: PMC7055530]. Central to the rapid proliferation of cancer cells is the ubiquitin-dependent proteasome pathway and its requirement for key cellular processes such as cell cycle regulation, cell differentiation, and apoptosis. Thus, the effect on proteasomes to inhibit the rapid proliferation of cancer cells has previously been considered as a therapeutic strategy. Important here is the effect of blocking the proteasomal degradation of ubiquitinated proapoptotic proteins and, apparently, enhancing the apoptosis of cancer cells, presumably due to their accumulation, as seen in the example of the proapoptotic BAX protein [Li B, Dou QP. Box degradation by the ubiquitin/proteasome-dependent pathway: involvement in tumor survival and progression. Proc Natl Acad Sci USA. 2000 Apr 11; 97(8):3850-5. doi: 10.1073/pnas.070047997. PMID: 10725400; PMCID: PMC18105]. Although the exact mechanisms of proteolytic degradation of the ubiquitinated BAX protein are not well understood, they may represent an alternative therapeutic strategy in cancer.

One of the groups of intracellular proteinases that are responsible for regulating the death and metastasis of tumor cells are lysosomal cysteine cathepsins, which belong to the superfamily of papain-like proteinases [Turk V, St oka V, Vasiljeva O, Renko M, Sun T, Turk B, Turk D. Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochim Biophys Acta. Jan 2012; 1824(1):68-88. doi: 10.1016/j.bbapap.2011.10.002. Epub 2011 Oct 12. PMID: 22024571; PMCID: PMC7105208; Soond SM, Kozhevnikova MV, Townsend PA, Zamyatnin AA Jr. Cysteine Cathepsin Protease Inhibition: An update on its Diagnostic, Prognostic and Therapeutic Potential in Cancer. Pharmaceuticals (Basel). 2019 Jun 11; 12(2):87. doi: 10.3390/ph12020087. PMID: 31212661; PMCID: PMC6630828; Soond SM, Kozhevnikova MV, Zamyatnin AA Jr. 'Patchiness' and basic cancer research: unraveling the proteases. cell cycle. 2019 Aug; 18(15): 1687-1701. doi: 10.1080/15384101.2019.1632639. Epub 2019 Jun 24. PMID: 31213124; PMCID: PMC6649598]. Although they are usually expressed and located in lysosomes, at the onset of the disease or due to a number of signals associated with the onset of the disease, some of these cathepsins are secreted into the extracellular matrix (extracellular matrix, ECM) [Rozhin J, Sameni M, Ziegler G, Sloane BF . Pericellular pH affects distribution and secretion of cathepsin B in malignant cells. Cancer Res. 1994 Dec 15; 54(24):6517-25. PMID: 7987851; Kramer L, Turk D, Turk B. The Future of Cysteine Cathepsins in Disease Management. Trends Pharmacol Sci. 2017 Oct; 38(10):873-898. doi: 10.1016/j.tips.2017.06.003. Epub 2017 Jun 28. PMID: 28668224; Reiser J, Adair B, Reinheckel T. Specialized roles for cysteine cathepsins in health and disease. J Clin Invest. 2010 Oct; 120(10):3421-31. doi: 10.1172/JCI42918. Epub 2010 Oct 1. PMID: 20921628; PMCID: PMC2947230], where they can easily cleave ECM components and thus promote tumor cell growth and spread. One such proteinase is cathepsin S, which may be a suitable candidate for targeted therapy [Soond SM, Kozhevnikova MV, Townsend PA, Zamyatnin AA Jr. Cysteine Cathepsin Protease Inhibition: An update on its Diagnostic, Prognostic and Therapeutic Potential in Cancer. Pharmaceuticals (Basel). 2019 Jun 11; 12(2):87. doi: 10.3390/ph12020087. PMID: 31212661; PMCID: PMC6630828; Rudzinska M, ParodiA, Soond SM, Vinarov AZ, Korolev DO, Morozov AO, Daglioglu C, Tutar Y, Zamyatnin AA Jr. The Role of Cysteine Cathepsins in Cancer Progression and Drug Resistance. Int J Mol Sci. 2019 Jul 23; 20(14):3602. doi: 10.3390/ijms20143602. PMID: 31340550; PMCID: PMC6678516]. Although the expression of cathepsin S in tissues is relatively limited, it has been noted that it is expressed under the influence of a number of physiological conditions associated with hypoxia at the site of the tumor or under conditions of chemotherapeutic exposure. Therefore, it can show its activity as an inducible factor, which, in particular, promotes tumor development and, possibly, resistance to chemotherapy (Soond, S.M.; Savvateeva, L.V.; Makarov, V.A.; Gorokhovets, N.V.; Townsend, P.A.; Zamyatnin, A.A., Jr. Cathepsin S Cleaves BAX as a Novel and Therapeutically Important Regulatory Mechanism for Apoptosis Pharmaceutics 2021, 13, 339). In addition, it was found that the expression of cathepsin S contributes to the development of cardiovascular diseases of atherosclerotic origin [Wu H, Du Q, Dai Q, Ge J, Cheng X. Cysteine Protease Cathepsins in Atherosclerotic Cardiovascular Diseases. J Atheroscler Thromb. 2018 Feb 1; 25(2):111-123. doi: 10.5551/jat.RV17016. Epub 2017 Oct 5. PMID: 28978867; PMCID: PMC5827079] and is also involved in protein degradation during MHC-II mediated tumor antigen presentation and differentiation of M1-M2 tumor macrophages [Yang M, Liu J, Shao J, Qin Y, Ji Q, Zhang X, Du J. Cathepsin S-mediated autophagic flux in tumor-associated macrophages accelerate tumor development by promoting M2 polarization. Mol Cancer. 2014 Mar 2; 13:43. doi: 10.1186/1476-4598-13-43. PMID: 24580730; PMCID: PMC4015740], which only emphasizes the diverse activity of cathepsin S in various normal and pathological conditions [Honey K, Rudensky AY. Lysosomal cysteine proteases regulate antigen presentation. Nat Rev Immunol. 2003 Jun; 3(6):472-82. doi: 10.1038/nri1110. PMID: 12776207].

The prior art known cytotoxic agents, in particular, Taxol and its therapeutic analogues [Wang LG, Liu XM, Kreis W, Budman DR. The effect of antimicrotubule agents on signal transduction pathways of apoptosis: a review. Cancer Chemother Pharmacol. 1999; 44(5):355-61. doi: 10.1007/s002800050989. PMID: 10501907; Weaver BA. How Taxol/paclitaxel kills cancer cells. Mol Biol Cell. 2014 Sep 15; 25(18):2677-81. doi: 10.1091/mbc.E14-04-0916. PMID: 25213191; PMCID: PMC4161504; Martins I, Raza SQ, Voisin L, Dakhli H, Allouch A, Law F, Sabino D, De Jong D, Thoreau M, Mintet E, Dugue D, Piacentini M, Gougeon ML, Jaulin F, Bertrand P, Brenner C, Ojcius DM, Kroemer G, Modjtahedi N, Deutsch E, Perfettini JL. Anticancer chemotherapy and radiotherapy trigger both non-cell-autonomous and cell-autonomous death. Cell Death Dis. 2018 Jun 18; 9(7):716. doi: 10.1038/s41419-018-0747-y. PMID: 29915308; PMCID: PMC6006149], which act by inhibiting the depolymerization of tubulin and thus cause a series of rapidly proliferating cancer cells to stop cycle and subsequently die by apoptosis. However, some cells may exhibit resistance to the lethal properties of such compounds, and this resistance is usually associated with advanced cancers that have an increased ability to metastasize tumors [Einzig AI, Gorowski E, Sasloff J, Wiernik PH. Phase II trial of taxol in patients with metastatic renal cell carcinoma. Cancer Invest. 1991;9(2): 133-6. doi: 10.3109/07357909109044223. PMID: 1677827; Sangrajrang S, Fellous A. Taxol resistance. Chemotherapy. 2000 Sep-Oct; 46(5):327-34. doi: 10.1159/000007306. PMID: 10965098].

To search for potential cathepsin-specific antagonists that may be particularly promising for cancer treatment, various peptide sequences are determined, on the basis of which the development of therapeutic agents is possible [Rudzinska M, Parodi A, Maslova VD, Efremov YM, Gorokhovets NV, Makarov VA, Popkov VA , Golovin AV, Zernii EY, Zamyatnin AA. Cysteine Cathepsins Inhibition Affects Their Expression and Human Renal Cancer Cell Phenotype. Cancers (Basel). 2020 May 21; 12(5):1310. doi: 10.3390/cancers12051310. PMID: 32455715; PMCID: PMC7281206, Soond SM, Kozhevnikova MV, Townsend PA, Zamyatnin AA Jr. Cysteine Cathepsin Protease Inhibition: An update on its Diagnostic, Prognostic and Therapeutic Potential in Cancer. Pharmaceuticals (Basel). 2019 Jun 11; 12(2):87. doi: 10.3390/ph12020087. PMID: 31212661; PMCID: PMC6630828]. In general, taking into account recent advances in the determination of substrate cleavage sites using screening of peptide libraries, a clearer picture of the mechanisms of regulation of specificity and degree of cathepsin-substrate interaction by inhibitors has emerged. Thus, synthetic peptides have recently been described (derivatives of fluoromethyl ketones or chloromethyl ketones of 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)), which have a high affinity for the catalytic triad of cysteine cathepsins and the ability to specifically inhibit the proteolytic activity of cysteine cathepsins [Rudzinska, M.; Parodi, A.; Maslova, V.D.; Efremov, Y.M.; Gorokhovets, N.V.; Makarov, V.A.; Popkov, V.A.; Golovin, A. V.; Zernii, E.Y.; Zamyatnin, A.A. Cysteine Cathepsins Inhibition Affects Their Expression and Human Renal Cancer Cell Phenotype. Cancers 2020, 12(5), 1310].

Cathepsins play a mutually exclusive role in development, as cathepsin S mediates differentiation of tumor-associated M1-M2 macrophages, while cathepsin C is required for tumor-induced N1-N2 differentiation of neutrophils [Yang M, Liu J, Shao J, Qin Y, Ji Q, Zhang X, Du J. Cathepsin S-mediated autophagic flux in tumor-associated macrophages accelerate tumor development by promoting M2 polarization. Mol Cancer. 2014 Mar 2; 13:43. doi: 10.1186/1476-4598-13-43. PMID: 24580730; PMCID: PMC4015740]. Therefore, targeting specific substrates to block their ability to act on cathepsins may represent an alternative approach.

Closest to the claimed peptide are "BH-3 mimetics" [Merino D, Kelly GL, Lessene G, Wei AH, Roberts A W, Strasser A. BH3-Mimetic Drugs: Blazing the Trail for New Cancer Medicines. cancer cell. 2018 Dec 10; 34(6):879-891. doi: 10.1016/j.ccell.2018.11.004. PMID: 30537511; Walensky L.D. Targeting BAX to drug death directly. Nat Chem Biol. 2019 Jul; 15(7):657-665. doi: 10.1038/s41589-019-0306-6. Epub 2019 Jun 17. PMID: 31209350], where peptide therapeutic antagonists are defined from protein interaction regions between pro-apoptotic and anti-apoptotic BH3 domain-containing proteins. For example, the therapeutic agent ABT-199 disrupts the inhibitory activity of Bcl-2 with the BAX protein for the treatment of leukemia, thereby allowing BAX to oligomerize and induce mitochondrial outer membrane permeability (MOMP) and cell death through activation of the intrinsic apoptosis pathway [Souers AJ, Leverson JD, Boghaert ER, Adder SL, Catron ND, Chen J, Dayton BD, Ding H, Enschede SH, Fairbrother WJ, Huang DC, Hymowitz SG, Jin S, Khaw SL, Kovar PJ, Lam LT, Lee J, Maecker HL, Marsh KC , Mason KD, Mitten MJ, Nimmer PM, Oleksijew A, Park CH, Park CM, Phillips DC, Roberts AW, Sampath D, Seymour JF, Smith ML, Sullivan GM, Tahir SK, Tse C, Wendt MD, Xiao Y, Xue JC, Zhang H, Humerickhouse RA, Rosenberg SH, Elmore SW. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat Med. Feb 2013; 19(2):202-8. doi: 10.1038/nm.3048. Epub 2013 Jan 6. PMID: 23291630].

However, ABT-199 as a selective inhibitor of BCL-2 is proven effective only against hematological malignant tumors, while inhibition of certain cathepsins that cleave proapoptotic proteins, such as BAX, can induce the death of malignant cells of various types and with the greatest effect.

Mazovec GD, Turk V, ReedJC, Myers RM, Salvesen GS, Turk B. Selective disruption oflysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem. 2004 Jan 30; 279(5):3578-87. doi: 10.1074/jbc.M308347200. Epub 2003 Oct 27. PMID: 14581476]. Поэтому необходимо обеспечить стабилизацию проапоптотических ВН3-белков (таких как ВАХ) посредством ингибирования катепсина, позволяя этим ВН3-белкам вызывать апоптоз в отсутствие ингибирующего действия антиапоптотических ВН3-белков (таких как Bcl-xl). Как правило для этого может потребоваться разработка, по крайней мере, двух различных терапевтических средств, где первый агент должен быть нацелен на катепсин (что позволяет стабилизировать ВАХ), второй - на ВАХ (или Bcl-2/Bcl-xl), тем самым нарушая их ингибирующее взаимодействие и позволяя преобладать проапоптотической активности ВАХ. Хотя этот подход действительно имеет хороший потенциал для индивидуального нацеливания белок-связывающих партнеров, недостатки заключаются в том, что такой подход потребует значительного количества времени для определения эффективности отдельных терапевтических средств и того, насколько эффективным может быть каждый из них при совместном действии. Следовательно, необходимо изучить альтернативные стратегии для разработки схемы, в которой может быть осуществлено нацеливание не только на катепсин, но и его производные проапоптотические субстраты (путем отделения его от антиапоптотического регулятора), которые в совокупности являются важными посредниками в гибели раковых клеток через апоптоз.Recently, there has been great interest in the identification of cathepsins capable of cleaving and inactivating proapoptotic BH3 proteins in order to identify those enzymes that can be therapeutically affected. Some cathepsins can cleave cytoplasmic -BAX, -BID, -Bcl-xl and -Mcl-xl [Cao X, Deng X, May WS. Cleavage of Box to p18 Box accelerates stress-induced apoptosis, and a cathepsin-like protease may rapidly degrade p18 Box. Blood. 2003 Oct 1; 102(7):2605-14. doi: 10.1182/blood-2003-01-0211. Epub 2003 Jun 19. PMID: 12816867; Appelqvist H, Johansson AC, Linderoth E, Johansson U, Antonsson B, Steinfeld R, Kagedal K, Ollinger K. Lysosome-mediated apoptosis is associated with cathepsin D-specific processing of bid at Phe24, Trp48, and Phe183. Ann Clinic Lab Sc. 2012 Summer; 42(3):231-42. PMID: 22964611; Cirman T, Oresic K, Mazovec GD, Turk V, ReedJC, Myers RM, Salvesen GS, Turk B. Selective disruption oflysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem. 2004 Jan 30; 279(5):3578-87. doi: 10.1074/jbc.M308347200. Epub 2003 Oct 27. PMID: 14581476; cirman T,

Mazovec GD, Turk V, ReedJC, Myers RM, Salvesen GS, Turk B. Selective disruption oflysosomes in HeLa cells triggers apoptosis mediated by cleavage of Bid by multiple papain-like lysosomal cathepsins. J Biol Chem. 2004 Jan 30; 279(5):3578-87. doi: 10.1074/jbc.M308347200. Epub 2003 Oct 27. PMID: 14581476]. Therefore, it is necessary to ensure the stabilization of pro-apoptotic BH3 proteins (such as BAX) by inhibiting cathepsin, allowing these BH3 proteins to induce apoptosis in the absence of the inhibitory effect of anti-apoptotic BH3 proteins (such as Bcl-xl). As a rule, this may require the development of at least two different therapeutic agents, where the first agent should be aimed at cathepsin (which allows you to stabilize the VAC), the second - at VAC (or Bcl-2 / Bcl-xl), thereby disrupting their inhibitory interaction and allowing the proapoptotic activity of VAC to prevail. While this approach does have good potential for individually targeting protein binding partners, the disadvantages are that it would take a significant amount of time to determine the effectiveness of individual therapeutic agents and how effective each could be when acting together. Therefore, alternative strategies need to be explored to develop a scheme that can target not only cathepsin but also its derivative proapoptotic substrates (by separating it from an antiapoptotic regulator), which together are important mediators in cancer cell death via apoptosis.

The technical problem to be solved by the invention is the search for a peptide sequence capable of inducing apoptosis in a tumor cell, as well as inhibiting the functional (proteolytic) activity of cathepsin S, with the prospect of using it in the development of antitumor therapeutic agents.

Disclosure of the essence of the invention

The technical result of the invention is the suppression of the activity of cathepsin S and the induction of apoptosis by inhibiting the interaction of the inhibitory protein Bcl-xl (or Bcl-2) with VAC, stabilizing the monomeric VAC, and also inhibiting cathepsin S-mediated proteolysis of the polyubiquitinated derivative of VAC (p120VAC).

The technical result is achieved by using a specific peptide compound containing the FFSFGGAL sequence selected from proapoptotic BH3-domain-containing proteins, characterized by the ability to induce apoptosis in a tumor cell.

The invention also includes a pharmaceutical composition for inducing apoptosis in a tumor cell, containing said peptide compound and a pharmaceutically acceptable carrier.

This compound can act not only on cathepsin S, but also on its subsequent pro-apoptotic/anti-apoptotic target protein complex, i.e. is a single therapeutic agent with dual specificity. The main advantage of such an agent is that it has the ability to specifically target more than one protein intermediate as part of a major regulatory protein complex specific for a signaling pathway that is key to cancer development and progression.

A particular peptide variant derived from Pit2 and having the sequence FFSFGGAL (hereinafter CS-PEP1), as well as a peptide derivative of the BH3 domain-containing protein sequence, are suitable for any therapeutic application that includes the action of cathepsin S (or other cathepsins), pro-apoptotic proteins, containing the BH3 domain, or anti-apoptotic proteins containing the BH3 domain, responsible for the regulation of apoptosis (Fig. 1).

Brief description of the drawings

The invention is illustrated by illustrations, where

in FIG. 1 shows a diagram of the inhibitory action of the proposed peptide (yellow circle). LMP - lysosomal membrane permeability; MOMP - permeability of the outer membrane of mitochondria;

in FIG. 2 presents experimental data demonstrating that cathepsin S mediates BAX cleavage in vitro and in mammalian cells;

in FIG. 3 shows experimental data demonstrating that CS-PEP1 inhibits cathepsin S-mediated BAX cleavage in vitro;

in FIG. 4 shows experimental data demonstrating that CS-PEP1 inhibits Bcl-xl cleavage but destabilizes it in vitro and in mammalian cells;

in FIG. 5 shows experimental data demonstrating that CS-PEP1 stabilizes the BAX protein when cathepsin S is expressed;

in FIG. Figure 6 presents experimental data demonstrating that CS-PEP1 stabilizes the level of endogenous BAX protein, incl. in the presence of cathepsin S expression;

in FIG. 7 shows experimental data demonstrating that treatment of cells with CS-PEP1 inhibits cathepsin S-mediated cleavage of the polyubiquitinated p120BAX BAX derivative;

in FIG. 8 shows experimental data demonstrating that CS-PEP1 induces apoptosis and caspase-3 activation.

Implementation of the invention

Below is a more detailed description of the implementation of the claimed invention, which does not limit the scope of the claims of the claimed invention, but demonstrates the possibility of carrying out the invention with the achievement of the claimed technical result.

Initially, it was determined that the proapoptotic BAX protein and the antiapoptotic Bcl-xl protein are substrates for cathepsin S. Cathepsins can also be regulated by human inhibitor proteins (cystatins), which prevent excessive activation of the undesirable proteolytic activity of cathepsins [Turk V, Stoka V, Vasiljeva O, Renko M, Sun T, Turk B, Turk D. Cysteine cathepsins: from structure, function and regulation to new frontiers. Biochim Biophys Acta. Jan 2012; 1824(1):68-88. doi: 10.1016/j.bbapap.2011.10.002. Epub 2011 Oct 12. PMID: 22024571; PMCID: PMC7105208].

Breuer M, Dauben H, Win J, Huesgen PF, Doehlemann G. A fungal substrate mimicking molecule suppresses plant immunity via an inter-kingdom conserved motif. Nat Commun. 2019 Apr 5; 10(1): 1576. doi: 10.1038/s41467-019-09472-8. PMID: 30952847; PMCID: РМС6450895]. С использованием платформы BLAST при поиске по базе данных NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome'). был идентифицирован пептид из аминокислотной последовательности ингибитора Pit2, который также присутствует в α-5 спиральной петле Bcl-xl, и имеет последовательность FFSFGGAL (обозначен CS-PEP1);. Эта петля α-5 антиапоптотического белка Bcl-xl взаимодействует с ВН3 доменом проапоптотического ВАХ и это взаимодействие является ключевым этапом, ответственным за Bcl-xl-опосредованное ингибирование белка ВАХ во время активации апоптоза [Renault ТТ, Dejean LM, Мапоп S. A brewing understanding of the regulation of Bax function by Bcl-xL and Bcl-2. Mech Ageing Dev. 2017 Jan; 161 (Pt B):201-210. doi: 10.1016/j.mad.2016.04.007. Epub 2016 Apr 23. PMID: 27112371].A peptide region has been identified that originates from a proteinase inhibitor that shares similarities with the anti-apoptotic protein Bcl-xl. For this purpose, a plant protein inhibitor of papain-like proteinases with the Pit2 sequence was used [Misas Villamil JC, Mueller AN, Demir F, Meyer U,

Breuer M, Dauben H, Win J, Huesgen PF, Doehlemann G. A fungal substrate mimicking molecule suppresses plant immunity via an inter-kingdom conserved motif. NatCommun. 2019 Apr 5; 10(1): 1576. doi: 10.1038/s41467-019-09472-8. PMID: 30952847; PMCID: PMC6450895]. Using the BLAST platform when searching the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome'). a peptide from the amino acid sequence of the Pit2 inhibitor was identified, which is also present in the α-5 helical loop of Bcl-xl, and has the sequence FFSFGGAL (designated CS-PEP1); This α-5 loop of the anti-apoptotic protein Bcl-xl interacts with the BH3 domain of the pro-apoptotic BAX and this interaction is a key step responsible for Bcl-xl-mediated inhibition of the BAX protein during apoptosis activation [Renault TT, Dejean LM, Mapop S. A brewing understanding of the regulation of Bax function by Bcl-xL and Bcl-2. Mech Aging Dev. Jan 2017; 161(PtB):201-210. doi: 10.1016/j.mad.2016.04.007. Epub 2016 Apr 23. PMID: 27112371].

The peptide was synthesized by Peptech Inc (St. Petersburg, Russian Federation) by solid-phase peptide synthesis, in which the peptide is obtained by combining various amino acids with each other, followed by chemical modification to introduce functional groups. Methods for the chemical synthesis of a peptide are widely known and well described using methods known in the art [US 6015881; Mergler et al., Tetrahedron Letters 29, 1988, pp. 4005-4008; Mergler et al., Tetrahedron Letters 29, 1988, pp. 4009-4012; Peptides, Chemistry and Biology, ed. Kamber et al., ESCOM, Leiden, 1992, pp. 525-526; Riniker et al., Tetrahedron Letters 49, 1993, pp. 9307-9320; Lloyd-Williams et al., Tetrahedron Letters 49, 1993, pp. 11065-11133, and Andersson et al., Biopolymers 55, 2000, ss. 227-250]. The purity of the product was determined using MALDI-TOF, after which the peptide was lyophilized for commercial use.

To work with this peptide, a 10 mM stock solution of the peptide in dimethyl sulfoxide (DMSO, cell culture grade (Sigma)) was prepared and stored at 4°C. Subsequent dilutions were made in DMSO and used directly to treat cell lines grown on special in vitro culture media or to add to cathepsin S in in vitro BAX/Bcl-xl protein digestion experiments at pH 5 or 7.

The properties of the peptide were evaluated in vitro by biochemical methods and on tumor cell lines.

In addition to VAC among proaptotic BH3-domain-containing proteins, Bak, Mtd/Bok, Bcl-xs [Kelekar A, Thompson SW. Bcl-2-family proteins: the role of the BH3 domain in apoptosis. Trends Cell Biol. 1998 Aug; 8(8):324-30. doi: 10.1016/s0962-8924(98)01321-x. PMID: 9704409]. In addition, the presented peptide sequence may have some amino acid substitutions that do not lead to significant changes in physicochemical properties and are verified using molecular docking (for example, serine/threonine, alanine/valine/leucine, isoleucine, serine/threonine, lysine/arginine, asparagine/glutamine, etc.).

The invention also includes pharmaceutical compositions using a CS-PEP1 peptide having the FFSFGGAL sequence, or peptides having amino acid substitutions in the CS-PEP1 sequences that do not significantly affect the properties of the peptides. Pharmaceutical compositions can be used in relation to diseases and disorders associated with impaired expression of cathepsin S and/or proteins of the BCL-2 family.

The pharmaceutical composition includes the CS-PEP1 peptide, or its modifications with amino acid substitutions, and any pharmaceutically acceptable carrier known from the prior art in the form of an inert non-toxic material that does not react with the active ingredient, and also ensures the delivery and penetration of the active ingredient into the target tissue. The carrier is selected according to physicochemical parameters (solubility, reactivity with respect to peptides, method and conditions for introducing the composition).

The present invention is illustrated by specific examples of implementation, demonstrating the possibility of achieving the desired technical result.

Example 1. Determination of the ability of cathepsin S to cleave BAX in vitro and in cells (Fig. 2A)

At present, the mechanisms of regulation of cathepsin S (CS) during BAX cleavage are unknown. To reveal this relationship, recombinant (derived from E. coli) biologically active CS was co-incubated with purified BAX protein (Abeam) and a dose-dependent cleavage of BAX was observed (Fig. 2A). Equal amounts of bacterially expressed BAX p21 protein were exposed to active CS at pH 7 and the BAX cleavage reaction was analyzed by Western blotting using anti-BAX antibodies (Abeam). The filters were then examined for CS detection (lower panels, SCB). 769P and HEK293 cells were co-transfected with pBAX-HA and pCS-FLAG expression plasmids (at increasing doses, μg) for 24 hours, prestimulated with vehicle (left panels) or 2 μM siramesine (right panels) for 1 hour; further, the expression levels of p21 BAX, CS, and actin were analyzed in cell extracts. In this case, a dose-dependent splitting of the VAC was also observed (FIGS. 2B and C). p21 BAX protein levels were analyzed under cycloheximide stimulation conditions (FIG. 2D) after cells were transfected with pBAX-HA (0.25 μg) or pCS-FLAG (0.75 μg) only for 24 hours. p21 BAX expression levels were quantified, standardized for actin expression levels in cells stimulated at T=0 h to T=6 h. Blue and red curves show p21 BAX or p21 BAX levels when co-expressed with CS (respectively) shown in black. Thus, the destabilizing effects of CS-flag expression on BAX-HA were also confirmed in mammalian cells, where the half-life of the BAX-HA protein was significantly reduced compared to BAX-only expression (Figure 2.D, graph).

As can be seen from FIG. 2, CS expression results in the degradation of the BAX protein in vitro and in mammalian cells.

Example 2 Evaluation of the inhibitory activity of CS-PEP1 on cathepsin S-mediated BAX cleavage in vitro (FIG. 3A)

In order to assess the inhibitory activity of CS-PEP1 on CS-mediated BAX cleavage, mammalian-derived immunoprecipitated BAX-HA protein (Fig. 3A) or bacterially expressed BAX (Fig. 3B) was co-incubated with active CS and decreasing doses. inhibitor of CS-PEP1. Equal amounts of immunoprecipitated p21 BAX-HA protein derived from 0.25 μg of BAX-HA expression in HEK293 cells were exposed to active CS with decreasing amounts of CS-PEP1 at pH 7, and p21 BAX (or CS) cleavage products were detected using Western -blotting with antibodies against BAX (Abeam) or CS (SCB, upper and middle panels). Substrate levels of p21 BAC were quantified, standardized against BAC (lane 2) and displayed in the bottom panel (FIG. 3B). BAX cleavage reactions at pH 7 were incubated in the presence of decreasing doses of CS-PEP1 and the reactions were analyzed for p21 BAX cleavage products (upper panel) and CS level (middle panel) using anti-BAX (Abcam) or anti-CS antibody (SCB). Substrate levels p21 VAC were quantified, standardized against VAC (lane 2) and displayed in the bottom panel. Quantitative data are presented as ±SEM and their significance (where p<0.05) is determined using a two-tailed Student's t test (*p<0.05, **p<0.01 and ***p<0.001). The p21 BAX cleavage reaction can be significantly slowed down in the presence of 10 µM-100 nM CS-PEP1 or 10 µM-1 mM CS-PEP1 for p21 BAX and BAX, respectively, in vitro

As can be seen from FIG. 3, CS-PEP1 inhibits cathepsin S-mediated BAX cleavage.

Example 3 Determination of the ability of CS-PEP1 to cleave and destabilize Bcl-xl in vitro and in mammalian cells (Fig. 4A)

In vitro CS-mediated Bcl-xl cleavage assays were performed at pH 7 and 5 (upper panel) using GST-Bcl-xl as substrate in the presence of 10 μM CS-PEP1 (or vehicle). Substrate levels were quantified as shown in the bottom panel (white bars at pH 7 and black bars at pH 5) and digestion products were quantified by Coomassie blue staining (FIG. 4B). HEK293 cells were stimulated with decreasing doses of CS-PEP1 in the presence of vehicle or 5 μg/ml Ras for 24 hours and analyzed for p21 BAX (Abeam), Caspase-3 (Abeam), Bcl-xl (Abcam), CS (Invitrogen) and actin (abcam) expression. Quantitative data are presented as ±SEM and their significance (where p<0.05) is determined using a two-tailed Student's t test (*p<0.05, **p<0.01 and ***p<0.001).

As can be seen from FIG. 4, CS-PEP1 exhibits inhibitory activity against CS-mediated Bcl-xl cleavage in vitro at pH 7 (FIG. 4A, lane 8) significantly better than at pH 5 (FIG. 4A, lane 7). Endogenous Bcl-xl protein levels also remained destabilized in HEK293 cells when cells were co-treated with paclitaxel and 50 μM-10 nM, suggesting that CS-PEP1 may interfere with the stabilization of Bcl-xl in the presence of conventional chemotherapeutic drugs that show poor killing efficacy. kidney cancer cells.

Example 4. Determination of the ability of CS-PEP1 to stabilize the BAX protein during the expression of cathepsin S (Fig. 5A)

769P cells were costimulated with vehicle or 5 μg/ml Ras, 10 μM zVAD-fmk and 10 μM CS-PEP1 for 24 hours. Soluble WCL was analyzed for the expression of endogenous p21 BAX (Abeam), CS (Abeam) and actin (Abeam). p21 BAX protein levels were quantified, standardized for actin expression, and represented by black bars as fold changes compared to vehicle alone stimulations (FIG. 5B). Endogenous p21 BAX, CS and actin levels were determined in lysates of HEK293 cells by Western blotting after 24 hours of co-stimulation with Pac (5 μg/ml) and CS-PEP1 (10 μM) (Fig. 5C). HEK293 cells co-transfected with pBAX-HA and pCS-FLAG or pBAX-HA and pCS-C25A-FLAG were stimulated with vehicle or 10 μM CS-PEP1 for 24 hours and cycloheximide for a further 6 hours. At the indicated time points, cells were lysed and cell lysates (WCL) were analyzed for p21 BAX, CS and actin expression using Western blotting (FIG. 5D). The expression levels for p21 BAX were quantified, standardized for actin and expressed relative to the levels observed at T=0 hour. BAX and CS co-expression in the presence of CS-PEP1 stimulation (blue) and p21 BAX with CS-C25A co-expression (red) are shown as trend lines (black curves). Quantitative data are presented as ±SEM and their significance (where p<0.05) is determined using a two-tailed Student's t test (*p<0.05, **p<0.01 and ***p<0.001).

An effect of CS-PEP1 in stabilizing the levels of expressed BAX was observed when BAX-HA was co-expressed with CS (FIG. 5A) or endogenous BAX protein in the presence of paclitaxel-induced endogenous CS (FIG. 5B). This was confirmed by observing similar levels of stability for BAX-HA co-expressed with CS-C25A-Flag and BAX-HA co-expressed with CS-Flag and treated with CS-PEP1 (Fig. 5C) over a six-hour course, and where the corresponding half-lives VAC proteins were nearly identical (Figure 5D).

Example 5. Stabilization of CS-PEP1 level of endogenous BAX protein in cells

CS-PEP1 stabilizes the level of endogenous BAX protein (including in the presence of cathepsin S expression) in 769P and HEK293 cells (Fig. 6). 769P and HEK293 cells were transfected with 1 μg empty expression plasmid pcDNA3.1+, pCS-FLAG or PCS-C25A-FLAG for 24 hours and then treated with 10 μM CS-PEP1 for an additional 24 hours. Cells were collected, lysates were prepared and analyzed for endogenous expression of BAX (anti-BAX), cathepsin S (using anti-CS antibodies) and β-actin (anti-β-actin).

In control experiments, CS expression destabilized endogenous BAC levels (first blot, penultimate lane), while dominant inhibitory expression of CS-C25A abolished this effect (first blot, lane 8). When using HEK293 cells, endogenous BAC levels were markedly increased by CS-PEP1 treatment (second blot, lane 6). When CS is expressed, BAX levels appear to be elevated (given that β-actin levels are lower than basal compared to control conditions (see lanes 3 and 4, second blot). In HEK293, PVLE treatment had no measurable effect on endogenous BAX protein levels, neither with CS expression, nor with dominant-inhibitory expression of C25A (third blot).In aggregate, these results indicate that CS-PEP1 stabilizes the level of BAC both when cells are treated with inhibitors only, and when CS is expressed in 769P cells.A similar pattern was observed in HEK293 cells.

6. Determination of the ability of CS-PEP1 to inhibit cathepsin S-mediated cleavage of the polyubiquitinated p120 BAX BAX derivative (Fig. 7)

HEK293 cells co-expressing CS-Flag or pCS-C25A-Flag with BAX and HA-ubiquitin were further treated for 24 hours with 10 μM CS-PEP1 for 6 hours and whole cell lysates immunoprecipitated with anti-HA antibodies were purified. Immune complexes were analyzed for the presence of the BAX protein by Western blotting with long-term (upper panel) and short-term exposure (lower panel). The red arrow indicates the polyubiquitinated VAC derivative, p120 VAC.

As can be seen from FIG. 7, treatment of cells with CS-PEP1 also results in accumulation of the 120 kDa polyubiquitinated BAX derivative (Example 6), especially in the presence of the inactive CS-C25A-Flag mutant, implying that CS may be involved in the proteolytic cleavage of ubiquitinated BAX and therefore , may play an important role in the proteasomal degradation of BAX protein levels.

Example 7 Determination of the Ability of CS-PEP1 to Induce Apoptosis and Caspase-3 Activation

HEK293 (Fig. 8A) or 769P (Fig. 8C) cells, respectively, were co-stimulated with CS-PEP1 (10 μM), hydrogen peroxide (HP, 5 μM), or paclitaxel (Pac, 5 μg/ml) for 24 hours; apoptotic cells were stained with TUNEL and their number was determined. Soluble whole cell lysates (WCL) obtained from cells treated in the same manner as in panels A and C were analyzed for caspase-3 (Casp-3) and actin expression (FIG. 8C). Caspase-3 expression levels were quantified and standardized for actin expression relative to cells stimulated with vehicle alone (FIG. 8D, graph). Quantitative data are presented as ±SEM and their significance (where p<0.05) is determined using a two-tailed Student's t test (*p<0.05, **p<0.01 and ***p<0.001).

Treatment with 10 μM CS-PEP1 of HEK293 or 769P cells induced their apoptosis by approximately 6-58% and 1-43%, respectively (Fig. 8A). CS-PEP1 can also mutually enhance the apoptosis-inducing effects of hydrogen peroxide or paclitaxel treatment of 769 cells from 11% to 93% and 55% to 64%, respectively (Fig. 8C). Detection of an intermediate caspase-3 with a mass of 18 kDa was observed as a control indicator of apoptosis activation (Fig. 8B, D). Thus, CS-PEP1 induces apoptosis and caspase-3 activation.

Thus, according to the results of the studies, it was revealed that the FFSFGGAL peptide has a number of valuable therapeutic properties, since it can enhance BAC-mediated apoptosis due to the following abilities:

1. Inhibit cathepsin S activity (in the same way that a Pit2 proteinase inhibitor inhibits plant papain-like proteinases).

2. Inhibit the interaction of the Bcl-xl/BAX inhibitory protein (due to the fact that it is derived from the Bcl-xl hydrophobic pocket that binds the BH3 domain of the BAX).

Box, Bak and beyond - mitochondrial performance in apoptosis. FEBS J. 2018 Feb; 285(3):416-431. doi: 10.1111/febs.14186. Epub 2017 Sep 4. PMID: 28755482]).3. Suppress BAX/BAX protein interactions and oligomer formation (due to being derived from the Bcl-xl hydrophobic pocket that binds BAX), thereby stabilizing the monomeric BAX (and which can still induce apoptosis

Box, Bak and beyond - mitochondrial performance in apoptosis. FEBS J. Feb 2018; 285(3):416-431. doi: 10.1111/febs.14186. Epub 2017 Sep 4. PMID: 28755482]).

4. Inhibit cathepsin S-mediated proteolysis of the polyubiquitinated BAX derivative (p120 BAX).

Claims (2)

1. The use of a peptide compound containing the amino acid sequence FFSFGGAL to suppress the activity of cathepsin S, which causes the induction of apoptosis in a tumor cell. 2. The use of a peptide compound containing the amino acid sequence FFSFGGAL for the preparation of a pharmaceutical composition for suppressing the activity of cathepsin S, which causes the induction of apoptosis in a tumor cell.

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