CN109879855B

CN109879855B - Inhibitor targeting PD-L1 molecule on surface of tumor cell and application thereof

Info

Publication number: CN109879855B
Application number: CN201910233853.2A
Authority: CN
Inventors: 周金明; 谢永丽; 崔香玲; 丁寄葳; 李晓宇; 岑山
Original assignee: Institute of Medicinal Biotechnology of CAMS
Current assignee: Institute of Medicinal Biotechnology of CAMS
Priority date: 2019-03-26
Filing date: 2019-03-26
Publication date: 2021-01-05
Anticipated expiration: 2039-03-26
Also published as: CN109879855A

Abstract

The invention provides an inhibitor targeting a tumor cell surface PD-L1 molecule and application thereof. According to the invention, an Odyssey On/In Cell Western Blot screening system is utilized to find that compounds Cobimetinib (GDC-0973, RG7420), AT7519, Lapatinib, LY2090314 and KU-60019 can show a good effect of inhibiting the expression of PD-L1 In different Cell experiments under the condition of low cytotoxicity, and the inhibition effect is verified In two different types of tumor cells. Therefore, the compounds (especially KU-60019) are expected to be developed into small molecule inhibitors for clinically inhibiting the expression of PD-L1, and the corresponding kinases are pharmacological targets for researching PD-1/PD-L1 signal pathways.

Description

Inhibitor targeting PD-L1 molecule on surface of tumor cell and application thereof

Technical Field

The invention relates to the field of tumor immunotherapy, in particular to an inhibitor targeting a tumor cell surface PD-L1 molecule and application thereof.

Background

In the immune regulation of the body, each checkpoint molecule plays a complex role in immune regulation, and the molecules are various and are commonly seen as PD-1, CTLA-4, IDO, TIM3 and the like. Among them, programmed death receptor (PD-1) has been the most focused in clinical studies at present and has been successfully applied in clinical studies, greatly driving the progress of tumor immunotherapy. PD-1 has two ligands, namely programmed death ligand 1(PD-L1) and programmed death receptor ligand 2(PD-L2), PD-L1 or PD-L2 is combined with PD-1, and antigen receptor signal transduction leads two tyrosine signal motifs in the cytoplasmic region of PD-1 to be phosphorylated, thereby activating a downstream signal pathway, finally destroying sugar metabolism of T cells and generation of signal factors such as IL-2 and the like, and leading the T cells to lose immune functions. Under normal conditions, the body prevents excessive activation of the immune system and maintains immune tolerance of the system to self-antigens by virtue of negative immune regulation of the PD-1/PD-L1/PD-L2 signaling pathway. However, overexpression of the checkpoint protein in this signaling pathway results in a co-inhibitory signal against the T Cell Receptor (TCR) or B Cell Receptor (BCR) cross-link and promotes pathological tolerance of the immune system. Over-expression of PD-L1/PD-L2 in the tumor microenvironment leads to the generation of immunosuppressive effects, so that the tumor cells under the conditions escape the monitoring of the immune system, and can rapidly proliferate and metastasize.

Drugs that block the PD-1/PD-L1/PD-L2 signaling pathway that are currently successfully used in clinical therapy are mainly antibody drugs, such as nivolumab and pembrolizumab (pembrolizumab) approved by the FDA in the united states for advanced melanoma at 12 and 9 months 2014, respectively. Meanwhile, since the checkpoint protein inhibitor is not directed to specific tumor cells per se, but to T lymphocytes in the tumor microenvironment, the research of the PD-1/PD-L1/PD-L2 checkpoint inhibitor is directed to other solid tumors besides melanoma. Such as the PD-1 blockers nivolumab and pembrolizumab were approved by the FDA in the united states for the treatment of advanced or metastatic non-small cell lung cancer at 3 months 2015. In addition, in 2016, the U.S. FDA re-approved altuzumab (atezolizumab) targeting PD-L1 for the treatment of a specific type of bladder cancer, and another PD-L1 inhibitor durvalumab (MEDI-4736, AstraZeneca/medimmunene) was approved by the FDA in 2017 for the treatment of PD-L1-positive metastatic urothelial bladder cancer. Currently, the Rogowski pharmaceutical attempts to use Altezumab and Durvalumab for the treatment of non-small cell lung cancer, and is already in clinical research. In addition, MPDL3280A for metastatic cell carcinoma of bladder or non-small cell lung cancer, BMS-936558 for non-squamous non-small cell lung cancer, and other monoclonal antibodies such as AMP-514 and pidilizumab have entered clinical research.

Although the monoclonal antibody targeting the PD-1/PD-L1/PD-L2 signal pathway is highly valued and breakthrough results are continuously generated, in recent years, few researchers develop the inhibitor targeting the PD-1/PD-L1/PD-L2 signal pathway to the field of traditional small molecule drugs, and the checkpoint protein small molecule inhibitor is more and more concerned due to the fact that the development cost is high, the requirements on transportation and storage conditions are strict, the stability of the antibody drug is poor, the preparation type is limited because the antibody drug cannot be orally taken, and the like. At present, small molecule inhibitors aiming at PD-1/PD-L1/PD-L2 signal paths are in the phase I clinical stage, but any small molecule compound is not approved to be on the market yet.

Kinase inhibitors have been widely used in the treatment of many cancers, and currently over 300 kinase inhibitors have been used clinically or in clinical studies. The ligand PD-L1 of PD-1 is abundantly expressed in various tumor cells. Therefore, if an effective screening scheme can be utilized to screen a compound capable of effectively inhibiting the tumor cell surface PD-L1 from a kinase inhibitor, a new candidate compound is provided for the research and development of a small molecular compound targeting a PD-1/PD-L1 signal pathway, and a new pharmacological target is provided for an immunotherapy means targeting the signal pathway, so that a more bright treatment prospect is brought to tumor patients.

Disclosure of Invention

The invention aims to provide an inhibitor targeting a tumor cell surface PD-L1 molecule and application thereof.

In order to achieve the object, in a first aspect, the invention provides an inhibitor or an inhibitor pharmaceutical composition targeting a tumor cell surface PD-L1 molecule, wherein an active ingredient of the inhibitor or the inhibitor pharmaceutical composition is selected from any one of compounds represented by formulas (1) to (5):

preferably, the active ingredient is a compound represented by formula (5).

In a second aspect, the invention provides an inhibitor or an inhibitor pharmaceutical composition targeting a PD-1/PD-L1 signaling pathway, wherein the effective component of the inhibitor or the inhibitor pharmaceutical composition is selected from any one of compounds shown in formulas (1) to (5). Preferably, the active ingredient is a compound represented by the formula (5).

In a third aspect, the invention provides any one of the following uses of the inhibitor or inhibitor pharmaceutical composition:

1) used for preparing the medicine for enhancing the targeting effect of the medicine for treating the tumor;

2) used for preparing the medicine for enhancing the anti-tumor effect;

3) is used for preparing a medicament for enhancing the blocking effect on the PD-L1 locus.

The combination drugs in the pharmaceutical composition of the present invention are used sequentially.

The tumor provided by the invention comprises human fibrosarcoma, non-small cell lung cancer, lymphoma, chronic myelogenous leukemia, acute lymphoblastic leukemia, breast cancer or melanoma.

Further, the inhibitor or the inhibitor pharmaceutical composition and pharmaceutically acceptable auxiliary materials are prepared into pharmaceutically acceptable dosage forms.

The object of the invention can be further achieved by the following technical measures.

The invention provides an effective screening scheme directly aiming at the expression of PD-L1 on the surface of a tumor cell membrane, so that a small molecular compound for inhibiting the expression of PD-L1 can be effectively screened out; further, compounds capable of inhibiting the expression of PD-L1 are sought from developed mature kinase inhibitors that are marketed or in the clinical stage, thereby providing new candidate compounds and new pharmacological targets for targeting the PD-1/PD-L1 signaling pathway.

The invention utilizes an Odyssey instrument produced by the American LI-COR company to establish a quantitative Odyssey On/In Cell Western Blot screening model for directly detecting the expression degree of PD-L1 On the surface of a tumor Cell membrane by optimizing conditions of Cell fixation, Cell punching, antibody type, antibody concentration, incubation mode, time and the like. In the model construction, firstly, a screening model is established by selecting a human meat cellulose tumor cell-HT-1080 cell. The cell has good cell adherence and reproductive growth capacity while highly expressing PD-L1, and greatly improves the screening efficiency. Secondly, the invention firstly uses the antibody of the PD-L1 protein extracellular region to detect the expression of the protein under the condition of keeping the integrity of the cell membrane, thereby avoiding the influence on the protein structure caused by cell pore-punching and ensuring the reliability of the detection result. Finally, the invention takes the beta-actin as the reference protein, and the result is presented by the ratio of the fluorescence value of the PD-L1 protein to the fluorescence value of the beta-actin protein, thereby greatly weakening the influence of an experimental system caused by cell density and toxicity of the screened drug and greatly reducing the false positive and false negative of the screening result.

The invention utilizes the established screening model to screen 430 Kinase inhibitors in a Kinase Inhibitor Library (96-well) -Z183593-100uL-L1200 compound Library of Selleck company. In primary screening and later-stage screening, the compounds Cobimetinib (GDC-0973, RG7420), AT7519, Lapatinib, LY2090314 and KU-60019 (corresponding to formulas (1) to (5) respectively) are found to be capable of obviously reducing the expression of PD-L1 on the surface of HT-1080 cell membrane. Then, the invention utilizes an Odyssey Western Blot experiment capable of quantifying to detect HT-1080 cells and non-small cell lung cancer-H-460 cells, and the results show that the 5 compounds can down-regulate the expression of PD-L1 in whole cells. The invention is further verified by flow cytometry, and the compounds Cobimetinib (GDC-0973, RG7420), AT7519, Lapatinib, LY2090314 and KU-60019 still can show good activity of inhibiting the expression of PD-L1.

Considering that the screened object of the invention is a kinase inhibitor which is marketed or in clinical research, namely the partial kinase inhibitor has a definite structure and partial common application, the compounds Cobimetinib (GDC-0973, RG7420), AT7519, Lapatinib, LY2090314 and KU-60019 which can inhibit the expression of PD-L1 obtained by the invention have great possibility of developing into a clinical PD-L1 small molecule inhibitor. In addition, the 5 compounds can show good inhibitory activity in two different tumor cells, and the expression of PD-L1 in various tumors is also shown. As mentioned above, the invention provides a new treatment scheme for tumor treatment because the combination of PD-1 and PD-L1 can be blocked by inhibiting the expression of PD-L1, thereby inhibiting the immune escape of the tumor.

Candidate compounds Cobimetinib (GDC-0973, RG7420), AT7519, Lapatinib, LY2090314 and KU-60019 discovered by the invention are inhibitors of the kinases MEK, CDK, EGFR, GSK-3 and ATM, respectively. The invention utilizes the same system to detect different kinase inhibitors, provides a reliable pharmacological target for the research and development of the PD-L1 small molecule inhibitor, and also provides a research basis for the regulation and control mechanism of the PD-1/PD-L1 signal channel in the tumor environment.

By the technical scheme, the invention at least has the following advantages and beneficial effects:

in the aspect of determination of a screening scheme, an Odyssey On/In Cell Western Blot screening system for directly detecting the expression level of PD-L1 On the surface of a tumor Cell membrane is successfully established by using an Odyssey instrument produced by LI-COR company In the United states. In the system, the fluorescence value of beta-actin protein is used as an internal reference, and the final screening result is presented through the ratio of the fluorescence values of PD-L1 protein and the internal reference protein, so that the false positive or false negative screening results caused by the difference of cell number and the toxicity of the screened drugs are eliminated in the screening process.

(II) the subject invention is directed to more mature kinase inhibitors that are already on the market or in clinical studies, and not to a completely new unknown compound library, thereby increasing the likelihood that the results from the screening will be useful in clinical drug development. Through rigorous screening, the invention discovers that the compounds Cobimetinib (GDC-0973, RG7420), AT7519, Lapatinib, LY2090314 and KU-60019 can show good effect of inhibiting the expression of PD-L1 in different cell experiments under the condition of low cytotoxicity, and the inhibition effect is verified in two different types of tumor cells. Therefore, the compounds (especially KU-60019) are expected to be developed into small molecule inhibitors for clinically inhibiting the expression of PD-L1, and the corresponding kinases are pharmacological targets for researching PD-1/PD-L1 signal pathways.

Drawings

FIG. 1a is a graph showing the expression level of PD-L1 in different types of tumor cells detected by Western Blot in example 1 of the present invention; FIG. 1b is a diagram showing the detection of PC-3 cells with low expression of PD-L1 and HT-1080 cells with high expression of PD-L1 by using Odyssey Western Blot in example 1 of the present invention; FIG. 1c shows the detection of PC-3 and HT-1080 expression levels using the Odyssey On/In Cell Western Blot; FIG. 1d shows the expression level of PD-L1 after the addition of a positive drug-inhibitory positive drug is Dinaciclib and the stimulatory positive drug is IFN-. gamma. -by the detection of Odyssey Western Blot; FIG. 1e is a graph of the expression level of PD-L1 after addition of positive compound detected by the Odyssey On/In Cell Western Blot.

FIG. 2a is a total scatter plot of kinase inhibitor screening in example 2 of the present invention; FIG. 2b is a summary of the screening at a final concentration of 10uM of compound; FIG. 2c is a summary of the screening at a final concentration of 1uM compound.

FIG. 3a is a graph of the fluorescence intensity of the sample in example 3 of the present invention using the Odyssey Western Blot in HT-1080 cells; FIG. 3b is a corresponding diagram of quantitative analysis.

FIG. 4a is a graph of the fluorescence intensity of H-460 cells using the Odyssey Western Blot in example 4 of the present invention; FIG. 4b is a corresponding diagram of quantitative analysis.

FIG. 5a is a diagram showing peaks obtained by detecting 21 screened compounds by flow cytometry in example 5 of the present invention; FIG. 5b is a corresponding quantitative analysis chart.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

Example 1 establishing Odyssey On/In Cell Western Blot model for high-throughput detection of expression level of PD-L1 On Cell membrane surface

1. Odyssey Western Blot experiment

Mixing 1.0X 10⁶DU-145, PC-3, 22RV1, HT-1080, H-1299, H-1975, H-460, A-549 and HeLa cell suspensions at 2 mL/well were plated in 6-well plates. The culture media used for DU-145, 22RV1 and 22RV1 cells are RPMI 1640 medium containing 10% Fetal Bovine Serum (FBS); the culture media used by the PC-3 cells are respectively F-12K culture media containing 10% Fetal Bovine Serum (FBS); the culture medium for HT-1080 cells is MEM culture medium containing 10% Fetal Bovine Serum (FBS); the culture media used for A-549 cells and HeLa cells are DMEM culture media containing 10% Fetal Bovine Serum (FBS) respectively; cells were incubated at 37 ℃ with carbon dioxide (5% CO)₂) Culturing in a constant temperature incubator. When the cells grew to 90% or more, the medium was aspirated, 80. mu.L of RIPA lysate was added to each well, the lysate was transferred to a 1.5ml EP tube and lysed on ice for 30 minutes, 20. mu.L of 5 Xprotein loading buffer was added to each tube, and the tube was metal-bathed at 100 ℃ for 30 minutes. SDS-PAGE gel electrophoresis separation, and Odyssey Western Blot to detect the expression level of PD-L1.

FIG. 1a is a graph showing the expression level of PD-L1 in different types of tumor cells detected by Western Blot in example 1 of the present invention. The test result shows that HT-1080 has the highest expression level of endogenous PD-L1 in the 9 tested tumor cells. In addition, the cells are found to have good adherence ability, high growth and propagation speed and simple culture conditions in the process of culturing the cells, so the cells are typical cells selected by a screening system.

2. Western Blot experiment for Odyssey On/In Cell

Mixing 1.0X 10⁵HT-1080 and PC-3 cell suspensions at a concentration of 200 uL/well were seeded into 96-well plates with a clear black frame from Corning, and cells were uniformly shaken and then oxidized at 37 deg.CAnd culturing in a carbon constant-temperature incubator for 24-48 hours. The culture medium for HT-1080 cells is MEM medium containing 10% Fetal Bovine Serum (FBS), and the culture medium for PC-3 cells is F-12K medium containing 10% Fetal Bovine Serum (FBS). When the cell density reaches more than 90% and the cell grows to the bottom of the 96-well plate, the culture solution in the 96-well plate is removed by a liquid moving machine, and 200uLPBS is added into each well to wash the residual culture medium on a shaking table for 5 minutes. After removal of PBS, 150uL of fresh fixation buffer (3.7% paraformaldehyde) was quickly added to fix the cells and incubated at room temperature for 20min without shaking. The immobilization buffer was removed by pipette, followed by addition of 200uL PBS per well and washing of the residual buffer on a shaker for 15 minutes, with fresh PBS every 5 minutes. After complete removal of the fixation buffer, 150uL of LI-COR Odyssey blocking solution was carefully added from the tube wall to each well using a pipette and incubated on a shaker at room temperature for 1.5 hours with slow shaking. After removal of the blocking solution, an antibody to PD-L1 (ab205921, Abcam) was added and incubated overnight at 4 ℃ with shaking. The next day the antibody was recovered from the overnight incubated 96-well plates and unbound antibody was washed with PBS on a shaker for 20 minutes, with fresh PBS changing every 5 minutes. Subsequently, 200uL of Triton elution buffer (10% Triton X-100) was added to each well and allowed to stand at room temperature for 20 minutes. The elution buffer was aspirated off with a pipette, and the beta-actin antibody was added and incubated on a shaker at room temperature for 2 hours. Unbound antibody was then washed with PBS on a shaker for 20 minutes, with fresh PBS changed every 5 minutes. After the PBS was blotted thoroughly, the 96-well plate was scanned with an Odyssey instrument.

Three parallel and systematic controls were set up for the experimental groups and statistical analyses were performed, of which<0.05,**P<0.01，***P<0.001 is referenced to the Dimethylsulfoxide (DMSO) group of each group. Experimental data on

Expressed and plotted using GraphPad Prism 5.0 and statistically analyzed. FIG. 1c shows the expression level of PC-3 and HT-1080 using the Odyssey On/In Cell Western Blot.

FIG. 1b is an Odyssey On/In Cell Western Blot experiment for detecting PC-3 cells with low expression of PD-L1 and HT-1080 cells with high expression of PD-L1 In example 1 of the present invention. FIG. 1c shows the expression level of PC-3 and HT-1080 using the Odyssey On/In Cell Western Blot. The combination test result shows that compared with PC-3 cells, HT-1080 cells can highly express PD-L1 protein at the level of whole cell lysate and cell membrane. Compared with the Odyssey Western Blot experiment, the Odyssey On/In Cell Western Blot experiment has the advantages of better data reproducibility, higher general screening quantity, more sensitive detection degree, better linearity and the like.

FIG. 1d shows the expression level of PD-L1 after the addition of a positive drug, namely inhibitory positive drug, Dinaciclib and promoting positive drug, namely IFN-gamma, by the detection of Odyssey Western Blot. The pretreatment method of the test is exactly the same as the Odyssey Western Blot test in example 1, and 2uL of the corresponding drug is added to each well when the cells are cultured to 80%, followed by 24-hour culture for treatment. The latter treatment was exactly the same as the Odyssey Western Blot experiment in example 1.

FIG. 1e is a graph of the expression level of PD-L1 after addition of positive compound detected by the Odyssey On/In Cell Western Blot. The pretreatment method of the test is completely the same as the Odyssey On/In Cell Western Blot test In example 1, and when the cells are cultured to 80%, 1uL of the corresponding drug is added into each well, and then the cells are cultured for 24 hours for treatment. The latter treatment was exactly the same as in example 1 (2).

Compared with the test results, the Odyssey On/In Cell Western Blot experiment has more remarkable advantages. Due to the special sensitivity and the characteristic of high-throughput screening, the method becomes the first choice drug screening system of the invention.

Example 2 results of screening 430 kinase inhibitors Using the Odyssey On/In Cell Western Blot screening System

Three parallel and systematic controls were set up for the experimental groups and statistical analyses were performed, of which<0.05,**P<0.01，***P<0.001 is referenced to the DMSO group of each group. Experimental data on

Expressed and plotted using GraphPad Prism 5.0 and statistically analyzed.

The pretreatment method of the test is completely the same as the Odyssey On/In Cell Western Blot test In example 1, and when the cells are cultured to 80%, 1uL of the corresponding drug is added into each well, and then the cells are cultured for 24 hours for treatment. The latter treatment was exactly the same as the Odyssey On/In Cell Western Blot experiment In example 1. FIG. 2a is a total scatter plot of the kinase inhibitor screening in example 2 of the present invention, i.e., the combination of all available data. According to the concentration of the screened drug, the invention screens four working concentrations of 10uM, 1uM, 100nM and 10nM to determine the pharmacological activity of the screened compound. FIG. 2b is a summary of the screening at a final concentration of 10uM of compound; FIG. 2c is a summary of the screening at a final concentration of 1uM compound. The test results show that in the present screening, 43 compounds and 59 compounds showed more than 20% of activity of inhibiting the expression of PD-L1 compared to the blank control group (DMSO group) at the screening concentrations of 1uM and 10uM, respectively.

Example 3 validation of the inhibitory Activity of the screened Compounds on PD-L1 in HT-1080 cells at the Whole cell level Using the Odyssey Western Blot assay

On the basis of primary screening and secondary screening, 28 compounds are continuously screened from the obtained compounds with inhibitory activity and are verified by an Odyssey Western Blot experiment to detect the change of the expression level of PD-L1 at the whole cell level. Experimental groups three independent experiments were performed, each with reference to the DMSO group of each group. Experimental data on

Expressed and plotted using GraphPad Prism 5.0 and statistically analyzed.

The experimental results showed that a total of 15 compounds showed an inhibitory effect of PD-L1 at the level of the whole cell in the results of the Odyssey Western Blot rescreening (FIGS. 3, a and b).

Example 4 validation of the inhibitory Activity of the Compounds obtained by screening against PD-L1 in H-460 cells at the Whole cell level Using the Odyssey Western Blot assay

On the basis of primary screening, secondary screening and HT-1080 cell Odyssey Western Blot experimental verification, the invention is to be based onThe 25 selected compounds are verified in an Odyssey Western Blot experiment in H-460 cells serving as non-small cell lung cancer cells to detect the influence of the compounds on the expression level of PD-L1 at the whole cell level of the non-small cell lung cancer. Experimental groups two independent experiments were performed, each with reference to the DMSO group of each group. Experimental data on

Expressed and plotted using GraphPad Prism 5.0 and statistically analyzed.

Experimental results show that compounds obtained by screening an Odyssey On/In Cell Western Blot screening system established by HT-1080 cells still show good effect of inhibiting the expression of PD-L1 In non-small Cell lung cancer cells H-460. In particular, the compounds Cobimetinib (GDC-0973, RG7420), AT7519, Lapatinib, LY2090314 and KU-60019 can obviously inhibit the expression of PD-L1 in H-460 cells (FIG. 4, a and b).

Example 5 screening of the Effect of the obtained Compounds on the expression level of PD-L1 on the surface of cell Membrane Using flow cytometry

Flow cytometry: mixing 1.0X 10⁶HT-1080 cell suspension (2 mL/well) was seeded in 6-well plates and the cells were incubated in a carbon dioxide incubator at 37 ℃. The culture medium for HT-1080 cells was MEM medium containing 10% Fetal Bovine Serum (FBS). When the cells grow to about 80%, 2uL of DMSO and other corresponding compounds are correspondingly added into each hole, and the cells are continuously cultured in a constant-temperature incubator for 24 hours. When the cells grew to 90% to confluency, the cell surface was washed with 1mL of PBS per well, followed by digestion of the cells with 0.25% pancreatin into PBS and rendering them into a single cell suspension. Single cell suspensions were counted and appropriate amounts of antibody were taken according to antibody incubation instructions and PE-direct PD-L1 antibody was incubated for 30 minutes on ice protected from light. Unbound antibody was then washed with PBS for 9 minutes, with fresh PBS changed every 3 minutes. The prepared flow-assay cell fluid was assayed by a Guava EasyCyte 8HT flow cytometer from Millipore. Three parallel and systematic controls were set up for the experimental groups and statistical analyses were performed, of which<0.05,**P<0.01，***P<0.001Reference is made to the DMSO groups of each group. Experimental data on

Expressed and plotted using GraphPad Prism 5.0 and statistically analyzed.

FIG. 5a is a diagram showing peaks obtained by flow cytometry on 21 screened compounds in example 5 of the present invention, and FIG. 5b is a diagram showing the corresponding quantitative analysis. The experimental results show that the compounds Cobimetinib (GDC-0973, RG7420), AT7519, Lapatinib, LY2090314 and KU-60019 show obvious activity of inhibiting the expression of PD-L1 in tumor cells in combination with the Odyssey Western Blot experiment in FIGS. 3 and 4.

The invention provides application of an Odyssey On/In Cell Western Blot system In quantitative and high-pass detection of expression level of PD-L1 On the surface of a tumor Cell membrane, and determines the effect of compounds Cobimetinib (GDC-0973, RG7420), AT7519, Lapatinib, LY2090314 and KU-60019 In inhibition of expression of PD-L1 On the surface of the tumor Cell membrane, so that the Odyssey On/In Cell Western Blot system is applied to development of a PD-L1 small-molecule inhibitor. The invention tests the inhibition effect of 430 kinase inhibitors On PD-L1 In tumor cells which are marketed or In clinical research by establishing a screening method for directly detecting the expression level of Cell membrane PD-L1 by Odyssey On/In Cell Western Blot, and finds that the compounds Cobimetinib (GDC-0973, RG7420), AT7519, Lapatinib, LY2090314 and KU-60019 have obvious inhibition effect On the expression of PD-L1. The 5 compounds discovered by the invention correspond to 5 different kinases, and therefore, a new idea is provided for the research of PD-1/PD-L1 signal pathways in a tumor microenvironment.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. Use of compound KU-60019 for the manufacture of a medicament for enhancing the blocking effect at the PD-L1 site, wherein compound KU-60019 has the structure: