KR20180056366A - Pharmaceutical compositions comprising tubulosine for preventing or treating cancers - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating cancer comprising tubulosine as an active ingredient. In the compositions of the present invention, Tubulosin strongly inhibits both structurally activated cytokine-induced JAK3 signaling by inhibiting the activity of JAK catalysis in leukemia and lymphoma cell lines, resulting in the survival and proliferation of active JAK-mediated cancer cells And the like.

Description

[0001] The present invention relates to pharmaceutical compositions comprising tubulosine for preventing or treating cancers,

The present invention relates to a pharmaceutical composition for preventing and treating cancer comprising tubulosin.

Cytokines and growth factors play important roles in regulating biological responses such as cell growth, differentiation, immune homeostasis, autoimmune and inflammatory diseases. One of the important signals regulating this biological process is the Janus family tyrosine kinases (JAKs) and their substrate transcription factors, substrate transcription factors and STAT (signal transducer and activator of transcription) proteins. Mammals encode four JAKs (JAK1, JAK2, JAK3, and TYK2) and seven STATs (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B, and STAT6), and many cytokines and growth factors encode JAK or STAT proteins Lt; / RTI > et al., Annu Rev Med. 2015; 66: 311-328; Springuel L et al., Haematologica 2015; 100: 1240-1253].

JAK3 is mainly expressed in hematopoietic cells and is a common gamma chain (γc) that is a cytokine receptor of interleukin (IL) -2, IL-4, IL-7, IL-9, IL- ) Subfamily, which implies that JAK3 plays an important role in T-cell development and immune system homeostasis. However, abnormal activation of JAK3 / STAT signaling results in hematopoietic malignancy. Gene therapy with autosomal recessive severe combined immunodeficiency (SCID) using hematopoietic stem cell transplantation increases the risk of T-cell leukemia through direct activation of the γc-mediated JAK3 / STAT5 signaling in human patient and mouse models. Leukemia germ cells in about 70% of patients with acute myeloid leukemia (AML) [Birkenkamp KU et al., Leukemia 2001; 15: 1923-1931], various cell lines derived from human blood cancers such as mantle-cell lymphoma [Yared MA et al., Arch Pathol Lab Med. 2005; 129: 990-996], Burkitt's lymphoma [Gee K et al., Cell Immunol. 2001; 211: 131-42), anaplastic large cell lymphoma (ALCL) [Lai R et al., Hum Pathol. 2005; 36: 939-944], and enteropathy-associated T-cell lymphoma [Malamut G et al., J Clin Invest. 2010; 120: 2131-2143], JAK3-mediated STAT signaling was continuously activated. In addition, in pre-B-cell leukemia mouse models established by loss of function of the tumor suppressor B cell linker (BLNK), an inhibitor of autocrine JAK3 / BKL, persistently activated JAK3 / STAT signaling appear. The expression of BLNK was completely lost or decreased rapidly in pre - B cell acute lymphoblastic leukemia (ALL). Somatic mutations of the JAK3 allele were found in patients with AML and acute megakaryocytic leukemia (AMKL) and in cell lines, and in Down syndrome acute lymphocytic and myeloid leukemia, T cell ALL, and cutaneous T cell lymphoma (CTCLs) in high-risk child ALL. In this case, patients were continuously activated by JAK3 signal by gain-of-function. These results suggest that an abnormally activated JAK3 signal contributes to a specific subtype of hematopoietic stem cells and that JAK3 is a therapeutic target for the treatment of patients with these diseases.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a composition capable of treating various diseases caused by abnormal activity of JAK3 (Janus family tyrosine kinases). Thus, structurally-based computerized database screening using the National Cancer Institute (NCI) diversity set compound was used to identify tubulosine (NSC131547) as a potent and selective JAK3 inhibitor. Tubulosin blocked the catalytic activity of JAK3 and strongly inhibited the constitutively-active and cytokine-induced JAK3 signaling in leukemia and lymphoma cell lines, inhibiting active JAK3-mediated tumor cell survival and proliferation.

Accordingly, the present inventors have completed the present invention by confirming that tubulosin (NSC131547) directly blocks the catalytic activity of JAK3 and is a small-molecule substance capable of treating various diseases caused by abnormal activity of JAK3 .

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing, ameliorating or treating cancer comprising tubulosine.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition comprising (a) a therapeutically effective amount of tubulosine represented by Formula 1; And (b) a pharmaceutically acceptable carrier. The pharmaceutical composition for preventing, ameliorating or treating cancer comprises:

(I)

The present inventors conducted a structure-based computerized database screening using a National Cancer Institute (NCI) diversity set compound to develop a composition capable of treating various diseases caused by the abnormal activity of JAK3 (Janus family tyrosine kinases) As a result, tubulosine (NSC131547) was identified as a potent and selective JAK3 inhibitor.

Tubulosin blocked the catalytic activity of JAK3 and strongly inhibited the constitutively-active and cytokine-induced JAK3 signaling in leukemia and lymphoma cell lines, inhibiting active JAK3-mediated tumor cell survival and proliferation. Thus, the present inventors have confirmed that tubulosin (NSC131547) directly blocks the catalytic activity of JAK3 and is a small-molecule substance capable of treating various diseases caused by abnormal activity of JAK3.

Activation of JAK3-mediated signaling in patients with mutations leads to immune pathologies such as severe combined immunodeficiency (SCID) and hematopoietic malignancy due to T cell development and diminished immune homeostasis. This evidence suggests that JAK3 is an attractive therapeutic target in the treatment of various human diseases, including hematopoietic malignancies.

Thus, in order to identify a new small molecule inhibitor of JAK3, structure-based virtual screening was performed using the 3D structure of the JAK3 kinase domain and the diversity set compound of the National Cancer Institute. Tubulosin (NSC131547) was found to directly block the catalytic activity of JAK3. Tubulosin binds mainly to the kinase domain of JAK3 in the JAK family, effectively inhibiting JAK3 mediated signaling at low concentrations. Tubulosin did not inhibit the activation of other tumor cell kinases in various cancer cells. Finally, we observed that tubulosin reduced cell survival and proliferation in cancer cells expressing persistently-active JAK3.

In the present invention, Tubulosin selectively inhibited the activity of JAK3, demonstrating that it is a potential candidate for a small-molecule candidate to treat various diseases caused by the abnormal activity of JAK3.

In one embodiment of the present invention, the composition of the present invention inhibits the catalytic activity of JAK3 (Janus Kinase 3).

The composition of the present invention can be administered orally or parenterally. In the case of parenteral administration, the composition can be administered through various routes such as intravenous infusion, subcutaneous infusion, muscle infusion, intraperitoneal infusion, and transdermal administration.

The cancer of the present invention can be used for the treatment of cancer such as blood cancer, breast cancer, brain cancer, neuroendocrine tumors, gastric cancer, lung cancer, prostate cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, , Bone cancer, skin cancer, thyroid cancer, pituitary cancer, and ureter cancer.

The blood cancer of the present invention is lymphoma, leukemia or myeloma.

As used herein, the term " lymphoma " refers to non-Hodgkins lymphoma (NHL); Diffuse large B-cell lymphoma (DLBCL); Follicular lymphoma (FL); Hodgkin's disease; Burkitt ' s Lymphoma; Cutaneous T cell lymphoma; Primary central nervous system lymphoma and lymphomatous metastases.

The T cell lymphoma of the present invention is selected from the group consisting of, but not limited to: lymphoblastic lymphomas occurring in primitive lymphoid progenitor cells from the thymus; Mature or peripheral T-cell neoplasms include T-cell prolymphocytic leukemia, T-cell granular lymphocytic leukemia; Aggressive NK-cell leukemia; Cutaneous T cell lymphoma; Adult T-cells associated with anaplastic large cell lymphoma, enteropathy-type T cell lymphoma, and human T-lymphotropic virus-1 (HTLV-1) (T-cell leukemia / lymphoma, angioimmunoblastic T-cell lymphoma, subcutaneous T-cell lymphoma, and paracortex in the first place) Peripheral T cell lymphoma that does not grow in true follicular pattern.

B-cell lymphoma of the present invention is selected from, but is not limited to: diffuse large B cell lymphoma (DLBCL), follicular lymphoma, MALT (mucosa-associated lymphatic (B) cell lymphoma, (B) lymphoma, (C) lymphoma, (C) lymphoma, (C) lymphoma, (C) lymphoma, , lymphoma, lymph node, lymph node, lymph node, lymph node, lymph node, lymph node, lymph node, lymph node, lymph node, lymph node, lymph node, Lymphomatoid granulomatosis, and AIDS-associated lymphoma.

According to the present invention, myeloma is multiple myeloma.

The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are those conventionally used in the field of manufacture and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. Such solid preparations can be prepared by adding to the pharmaceutical composition at least one excipient such as starch, calcium carbonate, sucrose, lactose , Gelatin and the like are mixed and formulated. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used.

Examples of the oral liquid preparation include suspensions, solutions, emulsions, syrups and the like. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, .

Examples of preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried agents, suppositories, and the like. Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Injectables may include conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers, preservatives, and the like.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to one embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-10000 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a pharmaceutical composition for preventing, ameliorating or treating cancer comprising tubulosine.

(b) tubulosin selectively inhibits JAK3 activity among JAK family members, so that it can be used as a therapeutic agent for treating various diseases (particularly, cancer) due to non-ideal JAK3 activity.

Figures 1a-1c. Structure - based virtual database screening model. 1a) The chemical structure of tubulosine (C ₂₉ H ₃₇ N ₃ O ₃ ; MW 475.6). 1b) Predictive binding model of tubulosin and JAK3 kinase domain (JAK3-JH1, PDB ID: 1YVJ). Only the side chain contacting within 3.5 Å of tubulosin was labeled. 1c) Overlap of ligands in the JAK3-JH1 complex. The structures of overlapping tubulosin (pink), AFN941 (cyan), CP-690550 (yellow), and CMP-6 (green) with JAK3 kinase domain were generated from the PDB files of 1YVJ, 3LXK and 3LXL, respectively. All structures were created with Pymol (pymol.sourceforge.net).
2a-2c. Tubulosin selectively inhibits JAK3 kinase activity in in vitro. 2a) Each JAK immunoprecipitate was pretreated with DMSO (vehicle), tubulocin (25, 50, 75, and 100 nM) or pan-JAK kinase inhibitor AG490 (150 μM) for 1 h, respectively. As a kinase reaction, His-tagged recombinant STAT3 alpha protein (2 mu g) and ATP (2 mu M) were added and reacted at 30 DEG C for 30 minutes. 2b) JAK3 immunoprecipitates were performed by ATP-dependent (2, 10, 50, 500, and 1000 [mu] M) bitroll kinase assays in the presence or absence of tubulosin (100 nM). The reaction products were confirmed by Western blotting using appropriate antibodies. 2c) In vitro kinase assays for four JAKs were performed with Merck Millipore's KinaseProfiler ™ Services. The IC ₅₀ values of tubulosin are listed in the table. Staurosporine (STS) was used as a positive control.
Figures 3a-3c. Tubulosin is an ATP-competitive kinase inhibitor against JAK3. 3a) JAK3 kinase assays were performed with ATP-concentration-dependent bits. 3b-3c) ATP-dependent EC ₅₀ (3b) and Km (3c) values of tubulosin for JAK3 kinase activity.
Figures 4a-4d. Tubulosin completely blocks intrinsically active JAK2 signaling in cancer cells. 4a-4d) human cancer cell lines L540 (4a), HDLM-2, MDA-MB-468, DU145 and A431 (4b), mouse leukemia cell lines BKO-84, BaF3 / JAK3V674A and BaF3 / TEL- (25, 50, 75, and 100 nM) or pan-JAK inhibitor AG490 (150 μM) were treated with DMSO, TEL-JAK2 (4d) for 24 hours. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as a loading control.
5a-5d. Tubulosin suppresses the JAK3 / STAT signal in cancer cells. 5a-5d) Toubulosin (100) was conjugated to Hodgkin's lymphoma cell line L540 (5a) and mouse leukemia cell line BKO-84 (5b), BaF3 / JAK3V674A (5c), and BaF3 / TEL- nM) for a certain period of time. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as a loading control.
6a-6b. High concentrations of tubulosin inhibit JAK1, JAK2, and / or TYK2-mediated signaling in cancer cells. 6a-6b) Hodgkin's lymphoma DMSO (vehicle), tubulosin or pan-JAK inhibitor AG490 (150 μM) was added to HDLM-2 cells (6a) and mouse leukemia BaF3 / TEL-JAK2 cells (6b) And reacted for 24 hours. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as a loading control.
Figures 7a-7c. Tubulosin blocks the IL-2-inducible JAK3 / STAT5 signal. (25, 50, 75, and 100 nM) or the pan-JAK inhibitor AG490 (150 μM) was starved for 16 hours, followed by treatment with DMSO Prolactin (PRL, 100 ng / mL, A) or IL-2 (100 ng / mL, B) was treated for 10 minutes. 7c) Human B-cell derived multiple myeloma U266 cells were treated with DMSO (vehicle), tubulosin (25, 50, 75, and 100 nM) or pan-JAK inhibitor AG490 , And IFN-α (1000 U / mL) for 30 minutes. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as a loading control.
8A-8B. Tubulosin does not affect the activation of other tumorigenic kinase signals in cancer cells. (50 and 100 nM) or a mixture of DMSO (vehicle), tubulosin (50 and 100 nM), or a mixture of human Hodgkin's lymphoma cell line L540 and HDLM-2 (8a) and human solid tumor cell lines MDA-MB-468, DU145 and A431 Pan-JAK inhibitor AG490 (150 μM) was treated and reacted for 24 hours. Western blotting was performed with whole cell lysates of the cells. GAPDH was used as a loading control.
Figures 9a-9b. Tubulosin selectively reduces the survival of cancer cells expressing constitutively-active JAK3. 9a-9b) were transfected with DMSO (vehicle), tubulosin (5'-deoxycytidine) in L540, BKO-84 and BaF3 / JAK3V674A cells (9a) and HDLM-2, MDA-MB-468, DU145 and BaF3 / TEL- 25, 50, and 100 nM) or pan-JAK inhibitor AG490 (150 μM). Western blotting was performed with whole cell lysates of the cells. GAPDH was used as a loading control. Cell viability was measured with WST-1 reagent and expressed as% control compared to DMSO (vehicle) -treated group. Results were expressed as mean ± standard deviation of three independent experiments (n = 3). * p < 0.05, ** p < 0.005.
10a-10b. Tubulosin inhibits the proliferation of cancer cells selectively expressing constitutively-active JAK3. 10a-10b) DMSO (vehicle), tubulo-neuropathicin (L-arginine) in L540, BKO-84 and BaF3 / JAK3V674A cells 10a and HDLM-2, MDA-MB-468, DU145 and BaF3 / TEL- 25, 50, and 100 nM) or pan-JAK inhibitor AG490 (150 μM). Cell proliferation was determined by counting viable cells using tryptophan blue assay. Results were expressed as mean ± standard deviation of three independent experiments (n = 3). * p < 0.05, ** p < 0.005.
Figure 11 is a schematic diagram illustrating that tubulosin selectively targets JAK3 and JAK3-mediated lower target signals.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

[Example]

Materials and Methods

1. Structure-Based Virtual Database Screening

To find small molecules that inhibit JAK2 activity by binding to the canase domain of JAK3 (JAK3-JH1), AutoDock ver. 4.2 was used and a virtual screening was performed with a set of NCI diversity of compounds. Docking is a two-step process.

In the first step, protein coordinates from the complex structure between JAK3-JH1 and its inhibitor staurosporine analog AFN941 were selected as templates. After removal of the ligand and solvent molecules, hydrogen atoms were added with AMBER software, which resulted in PDB2PQR-determined ionizable states in aspartic acid (Asp), glutamic acid (Glu), histidine (His) and lysine residues (Boggon TJ, Li Y, Manley PW, Eck MJ, Crystal structure of the Jak3 kinase domain in complex with a staurosporine analogue, Blood 2005; 106: 996-1002.)

In the second step, the inventors made detailed calculations with the candidates obtained in the first step. In addition, two JAK3-JH1 structure-complexes with CP-690550 (PDB ID: 3LXK) and CMP-6 (PDB ID: 3LXL) are included. 30 initial conformation of each small molecule by the AMBER package was generated and ensemble docking performed 90 times per compound.

The final structure was clustered based on structural similarity quantified as root mean square deviation (RMSD) between structures. And the center of the most dominant cluster was selected as representative. The values of 100 and 500,000 represent the parameter (ga_pop_size) for each number in the population and the maximum number of generations (ga_num_evals) for the generic algorithm in AutoDock. In-house written software and scripts automate all procedures. In order to understand the common and other characteristics of tubulocein binding to JAK1-JH1 and JAK2-JH1, the docking was repeated using the six procedures of JAK1-JH1 or JAK2-JH1 as templates in the same procedure. The PDB codes of the structure are 3EYG and 3EYH for JAK1-JH1 and JAK2-JH1 respectively; And 2B7A, 2W1I, 3E62, and 3FUP.

2. Tubulosine

Tubulosin (NSC131547) is one of the compounds identified in the NCI diversity set compounds and has been deposited with the Developmental Therapeutics Program (DTP) / NCI and provided to researchers for nonclinical purposes. Information on tubulogenesis and purity can be found on the DTP / NCI website.

3. Cell lines

The human Hodgkin's lymphoma cell lines HDLM-2 and L540 and the B-cell derived multiple myeloma cell line U266 were obtained from the German Collection of Microorganisms and Cell Cultures ; DSMZ, Germany). The human breast cancer cell line MDA-MB-468, the prostate cancer cell line DU145 and the melanoma cell line A431 were obtained from ATCC (American Type Culture Collection; Rockville, MD, USA) and cultured in DMEM medium containing 10% FBS. The Pre-B leukemia cell line BKO-84 was developed by Dr. Derived from BLNK - / - mice obtained from Daisuke Kitamura and consistently expressing the mutant JAK3 (BaF3 / JAK3V674A), TEL-JAK2 (BaF3 / TEL-JAK2) and TEL-JAK3 The pro-B cell line, BaF3, Hiroyuki Mano and Dr. Olivier A. Bernard, and the rat pre-T lymphoma cell line Nb2 was obtained from Dr. It was from Charles V. Clevenger.

4. Western blotting

Samples were separated by SDS-PAGE and transferred to nitrocellulose membrane. The membranes were treated with blocking buffer containing 5% steam milk, then treated with primary antibody against the target molecule and reacted overnight at 4 ° C. After washing the blot, the Horseradish peroxidase-conjugated secondary antibody was treated and reacted at room temperature for 2 hours. The signal was confirmed by ECL (enhanced chemiluminescence) reagent (SurModics Inc, Eden Prairie, MN, USA) Respectively. JAK2, phospho-JAK3 (Tyr980 / 981), JAK3, phospho-TYK2 (Tyr1054 / 1055), TYK2, phospho-STAT1 (Tyr701), STAT1, phospho-STAT3 (Tyr705), phospho-STAT5 (Tyr694), phospho-Src (Tyr416), Src, phospho-Lyn (Tyr507), Lyn, phospho-Akt 2, Thr202 / Tyr204), ERK1 / 2, PARP, caspase 3, caspase 9, Bcl-2, Bcl-xL, Mcl-1, survivin, Bax, Bad, p21, p27, RIP3, Beclin 1, LC3B and GAPDH Were purchased from Cell Signaling Technology (Cambridge, Mass., USA). STAT3 and STAT5 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

5. Cell proliferation and viability assays

Cells were plated in 96-well plates at 1 × 10 ⁴ cells / well and cultured until confluence of 70-80%. Cells were then treated with DMSO (vehicle), tubulocin, or AG490 (150 μM) at regular intervals. Cell viability was further allowed to proceed for 3 hours at 37 ° C. After addition of 10 μL assay reagent (EZ-CyTox Enhanced Cell Viability Assay Reagent, Daeil Lab Service, Seoul, Korea) USA) at 450 nm. On the other hand, viable cells were counted through trypan blue analysis and cell proliferation was measured.

6. In vitro kinase assay.

Each protein of the JAK family member was obtained by immunoprecipitation with a specific primary antibody and the recombinant His-tagged STAT3a protein was used as a substrate for the in vitro kinase assays. JAKs in vitro kinase assays were performed in a conventional manner (Kim BH et al., MS-1020 is a novel small molecule that selectively inhibits JAK3 activity. Br J Haematol. 2010; 148: 132-143 / NSC114792, a novel small molecule identified through structure-based computational database screening, selectively inhibits JAK3. Mol Cancer 2010; 9: 36.). On ice, HDLM-2 or L540 cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.4, 500 mM NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerophosphate, 1 mM DTT , 300 [mu] M Na3VO4, 1 mM PMSF and phosphatase inhibitor cocktail). The cell lysates were pre-cleared with protein A / G-sepharose at 4 ° C for 2 hours and the anti-JAK1, anti-JAK2, anti-JAK3, or anti-TYK2 antibodies were incubated overnight at 4 ° C lt; / RTI > Immune complexes were precipitated with protein A / G-sepharose beads. The precipitate was washed twice with kinase buffer (25 mM Tris / HCl, pH 7.5, 20 mM β-glycerophosphate, 10 mM MgCl 2, 2 mM DTT, 1 mM Na 3 VO 4, and protease inhibitor cocktail). For in vitro JAK kinase assays, immune complexes were mixed with DMSO (vehicle), tubulocin or pan-JAK inhibitor AG490 (150 μM) and reacted at 30 ° C for 1 hour. The kinase reaction occurred with the addition of His-tagged STAT3α protein (2 μg) at 0 ° C for 30 min with or without added ATP (2-1000 μM). SDS-PAGE was performed as a reaction product, and antibodies against phospho-STAT3, STAT3, JAK1, JAK2, JAK3, or TYK2 were used.

Other in vitro kinase assays were performed using Merck Millipore's KinaseProfiler ™ Services. Each IC ₅₀ value of the JAK family member was determined by performing a Millipore's standard radiometric assay at ATP (10 mM) and various concentrations of tubulin. The Ki and Km values of JAK3 were determined at various concentrations of tubulin and ATP. All kinase assays were performed twice with vehicle and acid blank controls, expressed as a percentage of vehicle negative control activity. The average activity (% control) of tubulosin was plotted against the compound concentration and the Ki value was calculated by linear regression y-intercept of the data according to the Cheng and Prusoff equation. The enzyme specific activity (U / mg) measured without tubulin was plotted according to ATP concentration and Km (app) for ATP was calculated by substituting into the Michaelis-Menten equation.

7. Statistics

Data were presented as means ± SD (SD), and statistical differences were determined using one-way analysis of variance and Dunnett's multiple comparison tests. Statistical differences were significant at p <0.05.

Experiment result

1. Identification of tubulosin as a JAK inhibitor through structure-based virtual screening

All JAK family members consist of seven Janus homology (JH) domains that share important structural homology. Of these domains, JH1 is a kinase domain and is important for JAK catalytic activity. To identify novel small molecules that inhibit JAK activity, AutoDock Version 4.2 for the kinase domain of JAK3 (JAK3-JH1) was used and virtual screening was performed with NCI diversity set compounds. Ensemble docking with a variety of early ligand conformers has improved the reliability of AutoDock. The results were superior to those obtained by simply increasing the number of runs with the same con- fomer, in terms of accuracy and energy.

Docking with existing molecules showed encouraging performance. Initially, docking was attempted with three small molecules AFN941, CP-690550, and CMP-6, which complex with JAK3-JH1. Each small molecule was docked in three coordinates, JAK3-JH1-1YVJ, 3LXK, and 3LXL. After clustering, the most populated clusters showed the lowest energy. Representative clusters in the best clusters showed a structure similar to that originally found in X-ray structures within a 1.0 Å RMSD value. It is noteworthy that the starting conformer of each molecule can be obtained from the 2D chemical structure of the public database (pubchem.ncbi.nlm.nih.gov) rather than the X-ray structure.

The model structure of tubulosin in the complex with JAK3-JH1 (FIG. 1A) is shown in FIG. 1A as Val-812, Ala-829, Glu-847, Met-878, Leu-881, Leu-932, and Asp-943 It was found to be in contact with the side chain atoms. The binding free energies of AFN941, CP-690550 and CMP-6 were -10.64, -9.23 and -10.32 kcal / mol, respectively, while the AutoDock-calculated binding free energy of JAK3-JH1 and tubulosin was -11.79 kcal / (Fig. 1C). The binding mode is similar to AFN941, CP-690550, and CMP-6 in the two-OCH ₃ surrounding regions, but there is a difference in the region contacting Asp-847.

2. Tubulosin directly blocks JAK3 kinase activity by binding to the ATP-binding site

The seven JH domains of JAKs have important structural homology. Based on this similarity, an in vitro kinase assay using immunoprecipitation of each JAK and recombinant STAT3a protein was performed to determine the specificity of tubulosin for JAK3 and / or other JAK3. JAK immunoprecipitates were obtained from cell lysates of Hodgkin's lymphoma HDLM-2 or L540 cells. Each JAK is activated in these cells. Each immunoprecipitate was reacted with the recombinant STAT3α protein under a tubulogenin concentration.

As a result, as in FIG. 2A, the STAT3a protein was efficiently phosphorylated by all JAK immunoreactive precipitants in the absence of tubuloin. This means that the immunoprecipitates have kinase activity. With AutoDock modeling, JAK3 kinase activity was effectively inhibited in the presence of tubulosine, but up to 100 nM of tubulosin did not affect other JAK activity (Fig. 2a). pan-JAK inhibitor AG490 blocked the kinase activity of all JAKs. Interestingly, JAK3 kinase activity is progressively decreased with ATP concentration (Figs. 2B and 3A). This means that Tubulosin is an ATP-competitive JAK3 inhibitor.

In order to determine the IC ₅₀ value of tubulosin for all JAKs, an in vitro kinase assay was performed using KinaseProfiler ™ Services. Tubulosin effectively inhibited JAK3 kinase activity with an IC _{50 of} 4.81, the inhibitory activity was reduced by ATP in a concentration-dependent manner, and the EC ₅₀ value was 2.38 μM. Although kinase activity of other JAKs was inhibited, it was lower than that of JAK3. The IC ₅₀ values of JAK1, JAK2, and TYK2 were 18.42, 25.67, and 22.69 nM, respectively (Figs. 2c and 3b). These results indicate that Tubulosin is a potent selective JAK3 inhibitor. Ki and Km values indicating JAK3 kinase activity were measured, 6.3 ± 0.6 nM when added with tubulosin and 106.4 ± 5.7 μM with ATP (FIG. 3C). At this time, staurosporine (STS) was used as a positive control for ATP-competitive inhibitors against JAKs.

3. Tubulosin completely blocks continuously-activated JAK3 signaling

As a result of the foregoing, the selectivity of tubulosin for inhibition of JAK3 was confirmed. To assess this selectivity, we examined whether Tubulosin can inhibit activated JAK3 signaling in various cancer cell lines.

As a result, the JAK3 signal is constitutively-active in cell lines L540, BKO-84, BaF3 / JAK3V674A, and BaF3 / TEL-JAK3 and other JAKs are not active. Tubulosin inhibits tyrosine phosphorylation of JAK3 and its substrate STAT5 in a concentration-dependent manner (Figs. 4A and 4C).

To examine the JAK3-mediated signal suppression, the stomach cell line was treated with 100 nM tubulosin and observed over time.

As a result, tubulosin effectively inhibited tyrosine phosphorylation of JAK3 and its substrates STAT3 and STAT5 within 2 hours (FIGS. 5A to 5D).

Cell lines HDLM-2, MDA-MB-468, DU145, A431, and BaF3 / TEL-JAK2 have other JAKs-mediated signals active than JAK3. Together with an in vitro kinase assays, the cell line did not affect tyrosine phosphorylation of JAK1, JAK2 and STAT5 until reaching a concentration of tubulin 100 nM (Fig. 4b and 4d). pan-JAK inhibitor AG490 completely blocked tyrosine phosphorylation of all JAKs and STAT5 in all cell lines. HDLM-2 and BaF3 / TEL-JAK2 cells were treated with tubulin at concentrations ranging from 100 nM to 1 μM for 16 hours to determine the tubulin cytotoxicity that inhibited other JAKs. JAK1, JAK2, and TYK2, and their substrates STAT3 and STAT5, were inhibited by tubulosin over a concentration of 400 nM (Figs. 6A and 6B). These results indicate that tubulosin completely inhibits the JAK3-mediated signaling pathway.

4. Tubulosin blocks cytokine-induced JAK3 signaling

We tested the selectivity of tubulosin for JAK3 signaling inhibition by IL-2 or prolactin (PRL) stimulation in rat pre-T lymphoma Nb2 cells that were used for cytokine-dependent activity studies of JAK signals . Cells were treated with DMSO (vehicle), tubulosin or AG490 for 16 hours and then with IL-2 or PRL for 10 minutes.

As a result, the phosphorylation status of certain tyrosine residues in JAK2, JAK3 and STAT5 was difficult to confirm, and their phosphorylation level was significantly increased by IL-2 or PRL (FIGS. 7A and 7B). As expected, JAK3 and STAT5 phosphorylation almost completely disappeared by low-concentration tubulosin up to 25 nM in IL-2-stimulated cells (Fig. 7A). However, tubulosin slightly inhibited PRL-induced JAK2 and STAT5 phosphorylation to a concentration of 100 nM (Fig. 7B).

Thus, we examined whether tubulosin affects JAK1- and / or TYK-2-mediated signals by treatment with interferon (IFN) -α.

As a result, IFN-α-stimulation significantly increased tyrosine phosphorylation of JAK1, TYK2, STAT1 and STAT3 compared to cells not treated with IFN-α in multiple myeloma U266 cells. Tyrosine phosphorylation was not affected up to a tubulin concentration of 100 nM (Fig. 7C). Pan-JAK inhibitor AG490 non-selectively inhibited tyrosine phosphorylation of all JAKs, STAT3 and STAT5 in cells treated with IL-2-, PRL-, or IFN- ?. These results clearly indicate that Tubulosin has selectivity for JAK3 over other JAKs.

5. Tubulosin increases ERK activity but does not affect other tumorigenic signals

The present inventors have investigated whether tubulosin can affect signals of other tumorigenic factors such as the Src family of non-receptor tyrosine kinases, serine / threonine kinase Akt, and extracellular signal-regulated kinase (ERK).

As a result, tubulosin was detected at the active level of Src family tyrosine kinase (phospho-Lyn in L540 and HDLM-2 cells, and in MDA-MB-468, DU145, and A431 cells until reaching a concentration of 100 nM phospho-Src) (Figs. 8a and 8b, lanes 3 and 4). In addition, no significant changes could be observed in the level of Akt phosphorylation after tubulin treatment of cells (Figs. 8a and 8b, lanes 5 and 6). Interestingly, the level of phosphorylation of ERK1 / 2 was significantly increased only in L540 cells expressing constitutively-active JAK3, in a tubulosin concentration-dependent manner. On the other hand, it was not observed in cell lines in which other JAK family members were activated (Figs. 8A and 8B, lanes 7 and 8). STAT3 phosphorylation in all cell lines showed a positive control and was effectively inhibited by tubulosin treatment in L540 cells and not in other cell lines such as HDLM-2, MDA-MB-468, DU145, and A431 (Figs. 8A and 8B , lanes 1 and 2). As a result, it can be seen that tubulosin selectively inhibits JAK 3 activity and suppresses its lower target signal transmission.

6. Tubulosin inhibits the survival and proliferation of cancer cells expressing persistently-active JAK3

Activated JAK / STAT signaling is associated with increased survival and proliferation of cancer cells, and small molecule inhibitors targeting this signaling have been reported to inhibit cell survival in a variety of solid tumor and hematological malignant cells. These findings demonstrate the potential that tubulosin will only affect cell survival in cancer cells that display a persistently active JAK3 signal. To demonstrate this possibility, cell survival analysis was performed by treating DMSO (vehicle), tubulosin or AG490 at various times in various cancer cell lines.

As a result, tubulosin, in a time and concentration dependent manner, significantly reduced the survival of various cancer cells expressing a persistently-active JAK3 signal, not other JAKs-mediated signal activated cells (FIGS. 9a and 9b). Inhibition of cancer cell viability was confirmed by the pan-JAK inhibitor AG490 treatment, which significantly reduced cell survival in all cell lines.

Then, the number of cells was counted using the trypan blue assay method to confirm the growth rate of cancer cells.

As a result, similarly to cell survival, tubulosin effectively inhibited the proliferation of cancer cells showing a persistently-active JAK3 signal in a concentration- and time-dependent manner (Fig. 10A). However, the proliferation of other JAK-activated cancer cells was less affected by tubulosin (Fig. 10B). With the cell survival rate, AG490 effectively inhibited the proliferation of cancer cells in all cell lines. These results indicate that JAK3 plays an important role in the survival and proliferation of cancer cells expressing a persistently active JAK3 signal. Inhibition of the JAK3 signaling pathway by tubulosin results in simultaneous tumor cell survival and proliferation inhibition.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

(a) a therapeutically effective amount of tubulosine represented by the following formula 1; And (b) a pharmaceutically acceptable carrier. The pharmaceutical composition for preventing, ameliorating or treating cancer,
(I)

The method according to claim 1,
Wherein said composition inhibits the catalytic activity of JAK3 (Janus Kinase 3).
The method according to claim 1,
Wherein said composition is administered orally or parenterally.
The method of claim 3,
Wherein said composition is parenterally administered by intravenous, subcutaneous, intramuscular, intraperitoneal or transdermal administration.
The method according to claim 1,
Wherein said cancer is selected from the group consisting of blood cancer, breast cancer, brain cancer, neuroendocrine tumors, gastric cancer, lung cancer, prostate cancer, ovarian cancer, liver cancer, bronchial cancer, nasopharyngeal cancer, laryngeal cancer, pancreatic cancer, bladder cancer, , Skin cancer, thyroid cancer, parathyroid cancer, or urethral cancer.
6. The method of claim 5,
Wherein said blood cancer is lymphoma, leukemia or multiple myeloma.
The method according to claim 6,
The lymphoma is non-Hodgkins lymphoma (NHL); Diffuse large B-cell lymphoma (DLBCL); Follicular lymphoma (FL); Hodgkin's disease; Burkitt &apos; s Lymphoma; Cutaneous T cell lymphoma; A primary central nervous system lymphoma or lymphomatous metastases. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;

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