Application of Aurora A inhibitor and LUBAC inhibitor in combination for the preparation of drugs for treating neuroblastoma

Abstract

The present invention belongs to the field of neuroblastoma treatment, and specifically relates to the use of an Aurora A inhibitor and a LUBAC inhibitor in combination in the preparation of a drug for treating neuroblastoma. The present invention has found that the combination of a small molecule inhibitor targeting LUBAC and an Aurora A inhibitor can significantly inhibit MYCN The activity of expanded NB cells was enhanced, and the effect of drug combination was better than that of drug alone, but it had no significant effect on normal cells and MYCN The inhibitory effect on the viability of non-expanded NB cells is not significant and can be used as MYCN Targeted drugs for expanded NB.

Description

Technical Field

[0001] The present invention belongs to the field of neuroblastoma treatment, and particularly relates to the use of an Aurora A inhibitor and a LUBAC inhibitor in combination in the preparation of a drug for treating neuroblastoma. Background Art

[0002] Neuroblastoma (NB) is the malignant transformation of neural crest cells or chromaffin cells during development. It is the most common extracranial malignant solid tumor in childhood, accounting for 8-10% of all childhood tumors and a mortality rate of approximately 15%. Its characteristics include insidious onset, high malignancy, rapid progression, and a high risk of recurrence, earning it the nickname "the king of childhood tumors." Children with high-risk NB experience insidious onset, high malignancy, and rapid progression. The traditional "surgery plus chemoradiotherapy" approach offers limited benefits, with most patients developing refractory NB and a five-year survival rate of only approximately 50%.

[0003] MYCN The gene belongs to the MYC family and encodes the transcription factor N-Myc, which regulates important biological processes such as cell proliferation, differentiation and apoptosis by transcribing a series of downstream target genes. MYCN The gene is amplified in many tumors, such as neuroblastoma, retinoblastoma, lung cancer, kidney cancer, etc. Clinical studies have shown that MYCN Gene amplification accounts for about 40-50% of high-risk NB patients and is the gold standard for the diagnosis of high-risk NB. MYCN High expression or MYCN There is still a lack of targeted drugs for the treatment of amplified tumors. Due to the structural characteristics of N-MYC protein, it is not easily bound by small molecule drugs and is currently considered to be a "drug-free" protein, which seriously limits the therapeutic effect of high-risk NB. MYCN Gene encoding usually appears in nervous system tumors (neuroblastoma NB, medulloblastoma MB, etc.) or tumors with neuroendocrine characteristics (small cell lung cancer SCLC, neuroendocrine prostate cancer NEPC, Wilms tumor, etc.) Gene amplification or high expression promotes tumor occurrence and development and is negatively correlated with prognosis.

[0004] Currently, new antisense oligonucleotides can inhibit N-Myc protein degradation by targeting it. MYCN On the other hand, more and more scientists are focusing on the indirect targeting strategy of N-Myc protein in order to achieve the goal of MYCNTreatment of high-risk NB with amplification. Studies have found that LIN28B and Aurora A kinase can regulate the stability of N-Myc protein, and Aurora A inhibitors can inhibit N-Myc at the mouse level. MYCN However, the high toxicity of high-dose inhibitors has limited their efficacy in clinical trials. Therefore, finding specific and precise therapeutic targets and drugs is an important way to improve the treatment of high-risk NB. MYCN The cure rate and long-term survival rate of children with expanded high-risk NB, as well as the prerequisite for improving the overall prognosis of children with NB, are also issues that urgently need to be addressed in clinical treatment.

[0005] The LUBAC complex is a linear ubiquitination E3 enzyme, the only one reported to date. It belongs to a new class of ubiquitin ligases. Unlike lysine-linked branched ubiquitination, LUBAC catalyzes head-to-tail linear ubiquitination. The LUBAC complex consists of three members: HOIP, HOIL-1L, and SHARPIN. HOIP is the catalytic subunit, while HOIL-1L and SHARPIN are regulatory subunits. The ubiquitination catalyzed by the LUBAC complex is non-degradative. Since its discovery in 2006, research on the LUBAC complex's functions has primarily focused on regulating immune pathways. Multiple studies have reported LUBAC mutations in various immune disorders, and targeting LUBAC holds promise for the treatment of diseases caused by activating mutations or aberrant LUBAC expression. Thiolutin or HOIPIN 11a are small molecule inhibitors targeting LUBAC, and some studies have shown that they can inhibit the progression of myeloid hematologic malignancies, but no studies have been conducted on neuroblastoma. Summary of the Invention

[0006] The object of the present invention is to provide a use of an Aurora A inhibitor and a LUBAC inhibitor in combination for preparing a drug for treating neuroblastoma.

[0007] Preferably, the Aurora A inhibitor includes the Aurora A inhibitor MLN8237.

[0008] Preferably, the LUBAC inhibitor includes the LUBAC inhibitor Thiolutin and / or the LUBAC inhibitor HOIPIN11a.

[0009] Preferably, the neuroblastoma is MYCN Amplified neuroblastoma.

[0010] According to a specific embodiment of the present invention, a composition for treating neuroblastoma comprises an Aurora A inhibitor and a LUBAC inhibitor.

[0011] According to a specific embodiment of the present invention, in the composition for treating neuroblastoma, the Aurora A inhibitor includes the Aurora A inhibitor MLN8237.

[0012] According to a specific embodiment of the composition for treating neuroblastoma of the present invention, the Aurora A inhibitor has an effective concentration at the cellular level of 0.01-0.05 μM.

[0013] Preferably, the Aurora A inhibitor has an effective concentration at the cellular level of 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM or 0.05 μM.

[0014] According to a specific embodiment of the present invention, in the composition for treating neuroblastoma, the LUBAC inhibitor includes the LUBAC inhibitor Thiolutin and / or the LUBAC inhibitor HOIPIN 11a.

[0015] According to a specific embodiment of the composition for treating neuroblastoma of the present invention, the LUBAC inhibitor has an effective concentration of 0.01-100 μM at the cellular level.

[0016] Specifically, the LUBAC inhibitor Thiolutin has an effective concentration at the cellular level of 0.1-0.5 μM, or any value within the above range, such as 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, or other values ​​within the above range, which are not listed here one by one.

[0017] The LUBAC inhibitor HOIPIN 11a has an effective concentration at the cellular level of 30-100 μM, or any value within the above range, such as 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, or other values ​​within the above range, which are not listed here one by one.

[0018] When used in combination, the molar concentration of the LUBAC inhibitor Thiolutin and the Aurora A inhibitor MLN8237 is 1:5-15; preferably, the molar concentration of Thiolutin is 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times or 15 times that of MLN8237.

[0019] When used in combination, the molar concentration ratio of the LUBAC inhibitor HOIPIN 11a to the Aurora A inhibitor MLN8237 is 1200-1600:1. Preferably, the molar concentration of the LUBAC inhibitor HOIPIN 11a is 1200 times, 1300 times, 1400 times, 1500 times or 1600 times that of the Aurora A inhibitor MLN8237.

[0020] The method of using the above-mentioned drug includes administering an effective amount of the above-mentioned drug to a subject. The administration method can be oral administration, drip infusion, intravenous injection or local tissue injection, or other reasonable methods to a patient in need of treatment.

[0021] A pharmaceutically effective amount refers to a quantity sufficient to treat a disease at a reasonable benefit / risk ratio that can be obtained by applying a drug therapy. The level of an effective dose can be determined based on a number of factors, including the patient's disease type, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration, excretion rate, treatment cycle, concurrently used drugs, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as an independent therapeutic agent or in combination with other therapeutic agents. Furthermore, the composition of the present invention can be added to a typical therapeutic agent continuously or simultaneously, and the composition can be administered single or multiple times. It is important to consider all of the above factors and administer the minimum dose that produces the maximum effect without side effects, which can be determined by a physician based on the patient's condition, age, etc.

[0022] According to a specific embodiment of the present invention, the composition for treating neuroblastoma is MYCN Amplified neuroblastoma.

[0023] Beneficial effects of the present invention:

[0024] The present invention found that the use of small molecule inhibitors Thiolutin or HOIPIN 11a targeting LUBAC in combination with the inhibitor MLN8237 targeting Aurora A can not only reduce the concentration of the Aurora A inhibitor MLN8237, but also significantly inhibit MYCN The activity of expanded NB cells was enhanced, and the effect of drug combination was better than that of drug alone, but it had no significant effect on normal cells and MYCN The inhibitory effect on the viability of non-expanded NB cells is not significant and can be used as MYCN Targeted drugs for amplified NB. The Aurora A inhibitor and LUBAC inhibitor of the present invention are used in combination, breaking through the existing small molecule inhibitors targeting MYCN The disadvantage of amplifying NB cells is poor specificity and high toxicity to normal cells, while the advantage of this method is high specificity and few side effects on other cell types. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0026] Figure 1 Knockdown of the LUBAC complex component HOIP MYCN Effects on the clone formation of the expanded NB cell line SK-N-BE(2);

[0027] A. Results of clone formation experiments;

[0028] B. Western immunoblotting results.

[0029] Figure 2 The effects of different concentrations of MLN8237 and Thiolutin (0.2 μM) on MYCN Effect of amplified SK-N-BE(2) cell viability on the cell line.

[0030] Figure 3 Shows the effects of MLN8237 (0.02μM) combined with Thiolutin (0.2μM) on the cell density and status of three cell lines: SK-N-BE(2), SK-N-SH, and IMR90.

[0031] Figure 4 The results show that different concentrations of MLN8237 and HOIPIN 11a (30 μM) MYCN Effect of amplified SK-N-BE(2) cell viability on the cell line.

[0032] Figure 5 Shown are the effects of MLN8237 (0.02 μM) combined with HOIPIN 11a (30 μM) on the cell density and status of three cell lines: SK-N-BE(2), SK-N-SH, and IMR90. DETAILED DESCRIPTION

[0033] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0034] Aurora A inhibitors are a class of drugs that inhibit the activity of Aurora A kinase, a serine / threonine kinase that plays a key role in multiple stages of the cell cycle, including centrosome duplication, spindle assembly, and chromosome segregation. Aurora A inhibitors include the small molecule inhibitors alisertib (MLN8237) and tozasertib (VX-680).

[0035] LUBAC inhibitors are a class of substances that inhibit the activity of the LUBAC complex. LUBAC (linear ubiquitin chain assembly complex) is an important protein complex that is primarily responsible for the synthesis of linear ubiquitin chains within the cell. These linear ubiquitin chains play a key role in numerous cellular signaling pathways, including immune signaling, apoptosis regulation, and other important biological processes. Thiolutin is a biologically active compound and a class of antibiotics. It acts as a linear ubiquitin chain assembly complex (LUBAC) inhibitor. HOIPIN 11a is a selective, cell-permeable, and covalent inhibitor that primarily inhibits the activity of the RBR E3 ubiquitin ligase HOIP. The biochemical properties of this compound indicate that it can affect a range of cellular activities, including but not limited to protein ubiquitination, by inhibiting HOIP function.

[0036] Experiments involved in the present invention:

[0037] Crystal violet staining (using a 6-well plate as an example) includes the following steps:

[0038] (1) Open the fume hood in advance, preheat 4% paraformaldehyde to 37°C, and prepare crystal violet staining solution;

[0039] (2) In a fume hood, discard the cell culture medium and quickly and gently add 1 ml of 4% paraformaldehyde along the side of the well plate and treat at room temperature for 15 minutes to fix the cells;

[0040] (3) Discard the paraformaldehyde and add 1 ml of crystal violet staining solution, and stain at room temperature for 15 min;

[0041] (4) After discarding the crystal violet stain, rinse repeatedly with distilled water until there is no staining in the gaps between colonies and the blank areas at the bottom of the well plate.

[0042] Determining cell viability includes the following steps:

[0043] (1) Prepare the CellTiter-Glo Luminescent Cell Viability Assay (CTG) kit in advance and prepare the system according to the instructions in the dark.

[0044] (2) 96 h after adding drugs to the cells in the 96-well plate, measure the total volume of culture medium in the well with the tip of a pipette and aspirate part of the culture medium to leave 50 μl of culture medium in the well;

[0045] (3) Add 50 µl CTG to each well in the dark.

[0046] (4) Place the 96-well plate with CTG in a light-proof box and shake for 2 minutes, then let it stand at room temperature in the dark for 10 minutes.

[0047] The fluorescence intensity in each well was collected and analyzed using the Firefly program in the microplate reader.

[0048] Example 1

[0049] NB cells (SK-N-BE2) were constructed to stably knock down the catalytic subunit HOIP of the LUBAC complex. The experimental group was shHOIP and the control group was shCtrl.

[0050] The specific experimental steps are as follows:

[0051] (1) Lentivirus construction: The lentivirus backbone vector GV493 (element sequence: hU6-MCS-CBh-gcGFP-IRES-puromycin) was used to construct a lentivirus containing the targeted knockdown of HOIP. The sequence of the targeted HOIP is: 5'-GGTACTGGCGTGGTGTCAAGTTTAA-3'.

[0052] (2) Cell inoculation: Inoculate SK-N-BE(2) cell suspension in each well of a 6-well plate to a cell number of 5.2*10 5 indivual;

[0053] (3) Virus infection was performed on the second day after plating, and the virus infection multiplicity of infection MOI was 10;

[0054] (4) Change the medium 24 hours after infection;

[0055] (5) After 48 hours, 2 mg / ml puromycin was added and treated for three days, and the cells became a stable knockdown cell line.

[0056] The clone-forming ability of cells was observed using the possibility formation experiment.

[0057] like Figure 1As shown in middle A, stable knockdown of HOIP, the catalytic subunit of the LUBAC complex, inhibited MYCN The amplified NB cell line SK-N-BE(2) proliferated. Figure 1 The protein immunoblotting results of B showed that shHOIP could knock down GAPDH as the internal reference gene. MYCN HOIP expression in the amplified NB cell line SK-N-BE(2).

[0058] It can be seen that stable knockdown of HOIP leads to a significant decrease in the number of NB cell clones and a weakening of the cell clone formation ability.

[0059] Example 2

[0060] MYCN amplified neuroblastoma cell line SK-N-BE(2) cells were selected.

[0061] Day 1: 1*10 3 Cells were seeded into 96-well plates;

[0062] The next day: Replace the culture medium with new DMEM complete medium, and add different concentrations of small molecule inhibitors Aurora A inhibitor MLN8237 (0, 1, 5, 10, 50, 75, 150, 200 nM) and LUBAC inhibitor Thiolutin (0.2 μM) to the culture medium. Treat the cells for 96 hours with 3 replicates per group.

[0063] Day 6: Cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay Kit.

[0064] Figure 2 The test results of the kit are shown. The cells were treated with different concentrations of Aurora A inhibitor MLN8237 and LUBAC inhibitor Thiolutin (0.2 μM). MYCN After 96 hours of treatment, the viability of the amplified neuroblastoma cell line SK-N-BE(2) was significantly inhibited. After the addition of Thiolutin (0.2μM), the half-lethal concentration of MLN8237 was reduced from 17.38nM to 4.16nM. Under low concentration conditions of MLN8237, the combined use of Thiolutin and MLN8237 had a significantly stronger killing effect on cells than the use of MLN8237 alone.

[0065] Example 3

[0066] MYCN amplified neuroblastoma cell line SK-N-BE(2) cells were selected. MYCNNon-amplifying neuroblastoma cell line SK-N-SH cells, MYCN Three non-amplified normal cell lines: IMR90.

[0067] Day 1: 1*10 3 Cells were seeded into 96-well plates;

[0068] The next day: replace with new DMEM complete medium, and add DMSO or small molecule inhibitor Aurora A inhibitor MLN8237 (0.02 μM) and LUBAC inhibitor Thiolutin (0.2 μM) into the medium. Treat for 96 hours with 3 replicates per group.

[0069] Day 6: The cells were stained with crystal violet. The results showed the number of stained live cells. The cells were photographed under a microscope and the live cell area was calculated.

[0070] The results are as follows Figure 3 As shown, after 96 h of treatment with the Aurora A inhibitor MLN8237 (0.02 μM) and the LUBAC inhibitor Thiolutin (0.2 μM), MYCN The number of viable cells in the amplified neuroblastoma cell line SK-N-BE(2) was significantly reduced, while MYCN The killing ability of the non-amplified neuroblastoma cell line SK-N-SH is weak. MYCN The non-expanded normal cell line IMR90 cells had almost no killing effect.

[0071] Example 4

[0072] MYCN amplified neuroblastoma cell line SK-N-BE(2) cells were selected.

[0073] Day 1: 1*10 3 Cells were seeded into 96-well plates;

[0074] The next day: Replace the culture medium with new DMEM complete medium, and add different concentrations of small molecule inhibitors Aurora A inhibitor MLN8237 (0, 1, 5, 10, 50, 75, 150, 200 nM) and LUBAC inhibitor HOIPIN 11a (30 μM) to the culture medium. Treat the cells for 96 hours with 3 replicates per group.

[0075] Day 6: Cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay Kit.

[0076] Figure 4The results of the test kit are shown, and the cells were treated with the Aurora A inhibitor MLN8237 and the LUBAC inhibitor HOIPIN 11a (30 μM) in combination. MYCN After the amplified neuroblastoma cell line SK-N-BE(2), the viability of SK-N-BE(2) cells was significantly inhibited, and under low concentration conditions of MLN8237, the combined use of HOIPIN 11a and MLN8237 had a significantly stronger cell-killing effect than the use of MLN8237 alone.

[0077] After the addition of HOIPIN 11a (30 μM), the half-lethal concentration of MLN8237 decreased from 18.52 nM to 4.18 nM.

[0078] Example 5

[0079] MYCN amplified neuroblastoma cell line SK-N-BE(2) cells were selected. MYCN Non-amplifying neuroblastoma cell line SK-N-SH cells, MYCN Three non-amplified normal cell lines: IMR90.

[0080] Day 1: 1*10 3 Cells were seeded into 96-well plates;

[0081] The next day: replace with new DMEM complete medium, and add DMSO or small molecule inhibitor Aurora A inhibitor MLN8237 (0.02 μM) and LUBAC inhibitor HOIPIN 11a (30 μM) into the medium. Treat for 96 hours with 3 replicates per group.

[0082] Day 6: The cells were stained with crystal violet. The results showed the number of stained live cells. The cells were photographed under a microscope and the live cell area was calculated.

[0083] like Figure 5 As shown, after 96 h of treatment with the Aurora A inhibitor MLN8237 (0.02 μM) and the LUBAC inhibitor HOIPIN 11a (30 μM), MYCN The number of viable cells in the amplified neuroblastoma cell line SK-N-BE(2) was significantly reduced, while MYCN The killing ability of the non-amplified neuroblastoma cell line SK-N-SH is weak. MYCN The non-expanded normal cell line IMR90 cells had almost no killing effect.

[0084] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Application of Aurora A inhibitors and LUBAC inhibitors in the preparation of drugs for the treatment of neuroblastoma; The Aurora A inhibitor is the Aurora A inhibitor MLN8237; The LUBAC inhibitor is the LUBAC inhibitor Thiolutin and / or the LUBAC inhibitor HOIPIN 11a.

2. The use according to claim 1, characterized in that Neuroblastoma is MYCN Amplified neuroblastoma.

3. A composition for treating neuroblastoma, characterized in that: The composition consists of an Aurora A inhibitor and a LUBAC inhibitor, wherein the Aurora A inhibitor is the Aurora A inhibitor MLN8237, and the LUBAC inhibitor is the LUBAC inhibitor Thiolutin and / or the LUBAC inhibitor HOIPIN 11a.

4. The composition for treating neuroblastoma according to claim 3, wherein The Aurora A inhibitor has an effective concentration of 0.01-0.05 μM at the cellular level.

5. The composition for treating neuroblastoma according to claim 3, characterized in that The LUBAC inhibitor has an effective concentration of 0.01-100 μM at the cellular level.

6. The composition for treating neuroblastoma according to claim 3, characterized in that Neuroblastoma is MYCN Amplified neuroblastoma.

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