CN108078991B

CN108078991B - Application of Aurora kinase inhibitor in preparation of tumor microenvironment inhibition drug

Info

Publication number: CN108078991B
Application number: CN201711484814.7A
Authority: CN
Inventors: 龙梓洁; 刘强; 鲁桂; 王君丹; 龙亮
Original assignee: Third Affiliated Hospital Sun Yat Sen University
Current assignee: Third Affiliated Hospital Sun Yat Sen University
Priority date: 2017-12-29
Filing date: 2017-12-29
Publication date: 2021-02-19
Anticipated expiration: 2037-12-29
Also published as: CN108078991A

Abstract

The inventionProvides an application of an Aurora kinase inhibitor in the preparation of drugs for inhibiting tumor microenvironment, the Aurora kinase inhibitor comprises a compound shown as a formula (I), and pharmaceutically acceptable salts, solvates, polymorphs, tautomers or prodrugs of the compound,

the Aurora kinase inhibitor has the effects of inhibiting tumor microenvironment, overcoming drug resistance and later-stage tumor recurrence, and has wider prospect and more excellent treatment effect.

Description

Application of Aurora kinase inhibitor in preparation of tumor microenvironment inhibition drug

Technical Field

The invention relates to the field of Aurora kinase, in particular to application of an Aurora kinase inhibitor in preparing a drug for inhibiting a tumor microenvironment.

Background

Aurora a (AURKA) kinases belong to the family of threonine/serine kinases, which regulate complex biological processes, and play an important role in controlling centrosome maturation and division and spindle function. Furthermore, AURKA is an important element for maintaining the self-renewal of embryonic stem cells, and knockout of AURKA significantly attenuates the self-renewal of embryonic stem cells and induces cell differentiation.

More and more studies show that AURKA can be used as a molecular marker closely related to tumorigenesis and tumor progression. The abnormal high expression of AURKA is found in breast cancer, gastric cancer, liver cancer, esophageal cancer, lung cancer, pancreatic cancer, ovarian cancer, bladder cancer and blood system tumors, and the abnormal high expression indicates poor prognosis. Overexpression of AURKA kinase can promote tumor cell proliferation, inhibit apoptosis, and accelerate cell metastasis. Conversely, inhibition of AURKA kinase expression may inhibit the progression of the tumor. Another report proves that AURKA plays a key role in maintaining the dryness of leukemia, colorectal cancer and breast cancer, inhibits AURKA to obviously weaken the dryness function of tumors, and further prompts that AURKA plays an important role in the occurrence and development of tumors.

The bone marrow microenvironment is also known as the soil for hematopoietic stem cells. The concept of Niche, proposed in the last 70 th century, generally included: stromal stem cells, extracellular matrix, and various cytokines, among others. The characteristic structure of the bone marrow microenvironment is closely related to normal hematopoietic stem cell mobilization, homing, and normal hematopoietic progression. Recent studies suggest that the bone marrow microenvironment is critical in the development of leukemia. The three-dimensional effect of the bone marrow microenvironment provides the effect of resisting chemotherapy drugs for leukemia cells, is the basis for the survival of the leukemia stem cells, and has important significance for the survival and drug resistance of the leukemia stem cells.

Mesenchymal Stem Cells (MSCs) are stromal stem cells in the bone marrow microenvironment, which constitute two functional implications with extracellular substances (cytokines, calcium ions, etc.). On one hand, the mesenchymal stem cells provide physical barriers for leukemia cells to play a role in drug resistance protection, and on the other hand, the secreted cytokines interact with surface receptors of the leukemia cells to activate proliferation channels of the leukemia cells and even promote the trans-vascular endothelial migration of the leukemia cells. Meanwhile, the research finds that the surface receptor integrin beta of the leukemia cell is adhered to an extracellular matrix and then interacts with the extracellular matrix to activate related channels such as PI3K/AKT and the like to promote the survival of the leukemia cell, and CD44 molecules expressed on the surface of almost all leukemia cells interact with mesenchymal stem cells to mediate the homing of the leukemia cell. In addition, cytokines secreted by leukemia cells, such as IL-1 beta, TNF-alpha, and the like, inhibit the survival of normal hematopoietic stem cells, thereby facilitating the growth of leukemia stem cells. In the process of occurrence and development of leukemia cells, the normal bone marrow microenvironment is gradually transformed into a support environment suitable for the growth of the leukemia cells, and finally, the normal hematopoietic process is inhibited.

The traditional treatment means is usually only directed at leukemia cells, and the residual intramedullary abnormal hematopoietic environment provides support for relapse after leukemia and leukemia cell drug resistance, so that a new treatment target point is urgently needed to be found to reverse the interaction between the leukemia cells and the hematopoietic microenvironment, not only the leukemia cells are specifically inhibited, but also the physical adhesion and the cell factor link between the leukemia cells and the microenvironment can be cut off, and therefore, the prognosis and the survival quality of patients with leukemia are improved.

Disclosure of Invention

The invention mainly aims to provide application of an Aurora kinase inhibitor in preparation of a tumor microenvironment inhibition drug, so as to solve the problem of poor prognosis effect of a leukemia treatment drug in the prior art.

In order to achieve the above object, the present invention provides an application of an Aurora kinase inhibitor in the preparation of a drug for inhibiting tumor microenvironment, wherein the Aurora kinase inhibitor comprises a compound shown as a formula (I), a pharmaceutically acceptable salt, a solvate, a polymorph, a tautomer or a prodrug of the compound,

further, the types of tumors in the tumor microenvironment include solid tumors and/or hematologic tumors.

Further, the solid tumor is any one of breast cancer, gastric cancer, liver cancer, esophageal cancer, lung cancer, pancreatic cancer, ovarian cancer and bladder cancer.

Further, hematologic tumors include leukemia.

Further, the microenvironment of leukemia is the bone marrow microenvironment.

Further, the bone marrow microenvironment comprises mesenchymal stem cells.

The technical scheme of the invention provides application of an Aurora kinase inhibitor in preparing a tumor microenvironment inhibition drug, wherein the Aurora kinase inhibitor comprises a compound shown as a formula (I), and pharmaceutically acceptable salts, solvates, polymorphs, tautomers or prodrugs of the compound. The inhibitor has the effects of inhibiting tumor microenvironment, overcoming drug resistance and later-stage tumor recurrence, and has wider prospect and more excellent treatment effect.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate embodiments of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:

FIG. 1 shows graphs of inhibition of leukemia cell AURKA kinase activity and promotion of apoptosis by Compound PW 21;

figures 2a and 2b show graphs of compound PW21 promoting apoptosis;

figure 3 shows compound PW21 inhibits cell clonogenic potential map;

figures 4a and 4b show the map of compound PW21 promoting cell cycle arrest;

figures 5a, 5b and 5c show graphs of the intervention effect of compound PW21 on MSC in Acute Myeloid Leukemia (AML) patients;

figure 6 shows a graph of inhibition of MSC-induced leukemia cell proliferation by compound PW 21;

figures 7a and 7b show compounds PW21 inhibits MSC-induced clonogenic potential of leukemia cells;

figures 8a and 8b show that compound PW21 inhibits MSC binding to leukemic cells in a potency map;

figure 9 shows compound PW21 inhibits MSC-induced leukemia cell migration potential.

Detailed Description

It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail with reference to examples.

As mentioned in the background art, the drugs for leukemia therapy in the prior art are usually only directed to leukemia cells, and the residual intramedullary abnormal hematopoietic environment provides support for the relapse of leukemia and the drug resistance of leukemia cells, so that the problem of poor prognosis effect exists. In order to find new therapeutic targets for reversing the interaction between leukemic cells and hematopoietic microenvironments, not only specifically inhibiting leukemic cells, but more importantly, cutting off the physical adhesion and cytokine link with the microenvironment, thereby improving the prognosis and quality of life of leukemic patients, the inventors screened the benzopyrimidine compound of formula (1) (named PW21) from known compounds having Aurora kinase inhibitory activity,

the inventor further researches and discovers that the compound has the inhibitory activity of AURKA kinase, also has the function of reversing the interaction between leukemia cells and a hematopoietic microenvironment, indicates good application potential and can overcome the defects in the prior art. Experiments prove that compared with the pyrimidine derivative in the prior art (which cannot act on a microenvironment and overcome drug resistance at the same time), the benzopyrimidine derivative PW21 has the effects of inhibiting a tumor microenvironment, overcoming the drug resistance and later-stage tumor recurrence, and has a wider prospect and a more excellent treatment effect.

Accordingly, based on the above research results, in one exemplary embodiment of the present application, there is provided a use of an Aurora kinase inhibitor for the preparation of a medicament for inhibiting tumor microenvironment, the Aurora kinase inhibitor comprising a compound represented by formula (I), a pharmaceutically acceptable salt, solvate, polymorph, tautomer or prodrug thereof,

in a preferred embodiment, the tumor types in the tumor microenvironment comprise solid tumors and/or hematological tumors.

In a preferred embodiment, the solid tumor is any one of breast cancer, gastric cancer, liver cancer, esophageal cancer, lung cancer, pancreatic cancer, ovarian cancer and bladder cancer.

In a preferred embodiment, the hematologic tumor comprises leukemia.

In a preferred embodiment, the microenvironment of the leukemia is the bone marrow microenvironment.

In a preferred embodiment, the bone marrow microenvironment comprises mesenchymal stem cells.

The relevant active effects of compound PW21 of the present application are further illustrated by the following specific examples.

Example 1

Aurora kinase activity Assay was determined by the Caliper Mobility Shift Assay method. Sequentially diluting compound PW21 three times from 10 mu M to obtain 10 concentrations in total, adding Aurora kinase, FAM labeled polypeptide and ATP, reacting at 25 ℃ for 60 minutes, and adding stop solution to stop the reaction; finally reading the conversion rate data by using a Caliper, converting the conversion rate data into inhibition rate data, and calculating by using Xlfit statistical software to obtain an IC₅₀And (4) data. The results of example 1 are shown in table 1.

Table 1: aurora kinase inhibitory Activity of Compound PW21

Compound PW21	AURKA	AURKB
			IC₅₀(nM)	6.0	152

As shown in Table 1, PW21 inhibits AURKA IC₅₀AURKB inhibiting IC at 6.0nM₅₀At 152nM, PW21 was shown to have specifically targeted inhibitory activity against AURKA kinase in vitro.

Example 2

Compound PW21 was dissolved in DMSO (100mM) as a stock solution and diluted at the time of use. Leukemia cells HL60 are treated by respectively taking compound PW21 with different concentrations, the cells are collected after 48 hours, the expression of phosphorylated AURKA (T288) and apoptosis specific protein clear caspase-8 in the cells is detected by Western blot, and GAPDH is taken as an internal reference.

The method comprises the following specific steps: collecting the cells treated with the different concentrations of PW21, and fully lysing the cells; the mixture was centrifuged at 14000 ℃ for 15 minutes to obtain a supernatant. Protein content was determined by Bradford protein quantitation. A protein sample of 100 mu g is taken, subjected to SDS-PAGE electrophoresis, transferred to an NC membrane, and subjected to antibody incubation and chemiluminescence development. The result of Western blot detection is shown in FIG. 1.

As can be seen from figure 1, in leukemia cell HL60, the expression of phosphorylated AURKA is obviously reduced along with the increase of PW21 dosage, and the expression of apoptosis-specific protein cleared caspase-8 is increased along with the increase of PW21 concentration (a strip shown by an arrow), which proves that PW21 can specifically target and inhibit the activity of AURKA kinase in cells so as to promote the leukemia cell to generate apoptosis.

Example 3

Leukemia cells were treated with varying concentrations of compound PW 21. Cells were harvested after 48 hours, stained with annexinV/PI, and apoptotic cell populations were detected by flow cytometry. The results are shown in FIG. 2a (NB4 cells) and FIG. 2b (HL60 cells).

FIGS. 2a and 2b show that PW21 promotes apoptosis of leukemia cells, manifested as early apoptosis (AnnexinV), in leukemia cells NB4 and HL60⁺/PI^-Q3) and late apoptosis (AnnexinV)⁺/PI⁺Q2) was significantly increased.

Example 4

Compound PW21 inhibited the clonogenic capacity of leukemia cells. Leukemia cells HL60, NB4 and retinoic acid resistant NB4R2 were cultured in methylcellulose and treated with the compound PW21 at various concentrations. After 10 days, the counting of the colony formation was observed under a microscope.

The results of example 4 are shown in FIG. 3. In leukemia cells, 0.3 mu M of PW21 can effectively inhibit the clonogenic capacities of leukemia cells HL60, NB4 and leukemia drug-resistant cells NB4R2, and the result shows that the compound PW21 has the function of inhibiting the self-renewal of leukemia stem cells.

Example 5

Treating leukemia cells NB4 and HL60 with compound PW21 of different concentrations, respectively, collecting cells after 48 hours, fixing with 70% ethanol, staining with 50 μ g/mL PI, and performing cell cycle detection with flow cytometry.

The results of the tests on leukemia cells NB4 and HL60 are shown in FIG. 4a and FIG. 4b, respectively. In leukemia cells, PW21 can obviously promote the cell cycle of leukemia cells to be blocked in the G2/M phase, and a polyploid phenomenon appears, which shows that PW21 promotes the cell cycle to be blocked by inhibiting AURKA, and further inhibits the proliferation of cells.

Example 6

MSCs from normal bone marrow donors were isolated by density gradient centrifugation and their associated specific surface antigens CD90 and CD105 were detected by flow cytometry. Western blot detection of NF- κ B expression (n is 3) in normal and AML MSCs; meanwhile, the compound PW21 with different concentrations is adopted to treat AML MSC, and Western blot is used for detecting the expression of NF-kappa B in the primary AML MSC.

The results of example 6 are shown in fig. 5a, 5b and 5 c. FIG. 5a shows that MSC-specific antigens CD90 and CD105 were positive in bone marrow isolated from normal subjects, indicating successful MSC extraction, and subsequent MSC extraction from normal subjects and AML patients were performed in the same manner. As shown in fig. 5B, MSCs of AML highly expressed NF- κ B compared to normal bone marrow donors. As shown in FIG. 5c, after PW21 treatment, the expression of NF- κ B in AML MSCs was significantly decreased, indicating that PW21 has inhibitory effect on MSCs in leukemia microenvironment, superior to that of general chemotherapeutic drugs.

Example 7

Compound PW21 was dissolved in DMSO (100mM) as a stock solution, diluted at the time of use, and added to the culture. Conditioned Media (CM) of MSCs cultured with AML were collected, filtered through a microfiltration membrane having a pore size of 0.45 μ M, and used to culture leukemia cells NB4 and HL60, while each leukemia cell was treated with a compound PW21 at different concentrations, and a fluorescent dye CFSE (fluorescent dye labeled with living cells) was added at a final concentration of 0.5 μ M, and the fluorescence intensity value of CFSE was measured by a flow cytometer after 48 hours.

The results of example 7 are shown in FIG. 6. PW21 (concentration of 0 μ M, 0.6 μ M, 1.2 μ M) has obvious inhibitory effect on proliferation of NB4 cells; similarly, PW21 (at concentrations of 0. mu.M, 0.6. mu.M, 1.2. mu.M) had a dose-dependent inhibitory effect on the proliferation of HL60 cells; the results indicate that PW21 can inhibit MSC-induced proliferation of leukemia cells.

Example 8

Normal CM (hBM CM) and AML CM (AML BM CM) were used to culture leukemia cells NB4 and HL60, respectively, and colony-forming experiments were performed. Compound PW21 was added to each group simultaneously, and colony formation was counted after 10 days.

The results of example 8 are shown in fig. 7a and 7 b. As shown in fig. 7a, CM from AML patients maintained the self-renewal capacity of leukemia cells better than normal bone marrow MSC, which was shown by a large increase in the number of leukemia cell clones NB4, HL60 after long culture (hBM CM vs. AML BM CM, p < 0.001). After adding compound PW21(0, 0.15 μ M, 0.3 μ M), compound PW21 inhibited the support effect of CM on leukemia cells in AML patients (0 μ M vs.0.3 μ M, p <0.001), and showed a dose-dependent inhibitory effect with increasing PW21 concentration, as shown in FIG. 7 b.

Example 9

MSCs of normal and AML were cultured in 6-well plates 24 hours before the experiment, respectively. After 24 hours, NB4 and HL60 were inoculated for co-culture, and 5X 10 cells were inoculated per well⁵The cells were added simultaneously with different concentrations of compound PW21, PBS washed off suspended NB4 and HL60 cells in the supernatant after 48 hours, MSC in the culture plate was digested with 0.25% trypsin, the cell pellet was resuspended and CD45 antibody and counting beads (CountBright) were added^TMAbsolute Counting Beads) for 20 min, and detecting CD45 by flow cytometry⁺The number of beads, the results of which were evaluated as the absolute number of cells bound to MSC.

The results of example 9 are shown in FIGS. 8a and 8 b. With the increase of the compound PW21 concentration, the capability of MSC of physically binding to leukemia cells NB4 and HL60 is reduced (0 μ M vs.2.5 μ M, p is less than 0.01), which indicates that the protection effect of MSC as a physical barrier can be inhibited under the action of PW 21.

Example 10

Compound PW21 inhibited the migration ability of myeloid microenvironment MSC-induced leukemia cells. MSCs from normal and AML were cultured in 24-Transwell plates 24 hours prior to the experiment. After 24 hours, the upper chamber was inoculated with 10⁷cells/mL leukemia cells NB4 or HL 60100 μ L, with different concentrations of compound PW21 added. The number of leukemia cells in the lower Transwell plates was counted after 12 hours. The results of example 10 are shown in FIG. 9, and the Transwell experiment represents the migratory capacity of cells, i.e., the metastasis of leukemia cells in vivo. The experimental result shows that the compound PW21 can reduce the migration capacity of NB4 and HL60 of leukemia cells under the induction of cell factors secreted by MSC (0 mu M vs.1.2 mu M, p)<0.001) to further demonstrate that PW21 can inhibit the supportive effect of the microenvironment on leukemia cells.

From the above description, it can be seen that the above-described embodiments of the present invention achieve the following technical effects: the compound PW21 has the inhibitory activity of AURKA kinase, and also has the capability of reversing the interaction between leukemia cells and hematopoietic microenvironment, indicating good application potential. Therefore, compared with the pyrimidine compound with Aurora kinase inhibitory activity in the prior art (which cannot act on a microenvironment and overcome drug resistance at the same time), the benzopyrimidine derivative PW21 has the effects of inhibiting a tumor microenvironment, overcoming drug resistance and later-stage tumor recurrence, and has a wider prospect and a better treatment effect.

In addition, the pharmaceutically acceptable salt, solvate, polymorph, tautomer or prodrug of the compound has the same or highly similar original drug structure as the compound and also has the effect of inhibiting leukemia microenvironment.

Moreover, different types of tumors have the characteristics of metastasis and recurrence, which are related to the specific tumor microenvironment of various tumors. Activation of the AURKA-NF- κ B signalling pathway occurs in almost different tumor microenvironments, and it is expected from the above examples that the benzopyrimidine compounds and their modified drug forms of the present application can also inhibit migration or recurrence of the tumor by inhibiting the action of stromal stem cells and their secreted cytokines in the respective tumor cell microenvironments. The application of the drug in the aspect of preparing the drug for inhibiting the tumor microenvironment has broad spectrum and can be suitable for the microenvironments of all the existing tumor types.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of Aurora kinase inhibitor in the preparation of medicine for inhibiting acute myeloid leukemia is characterized in that the Aurora kinase inhibitor is a compound shown in formula (I) or a pharmaceutically acceptable salt of the compound,