CN117942331A - PD-1/PD-L1 small molecule inhibitor and application thereof - Google Patents

Abstract

The invention discloses a PD-1/PD-L1 small molecule inhibitor which comprises an active ingredient baicalein or a structural analogue thereof and is used for inhibiting the combination of PD-1 and PD-L1. In-vitro and in-vivo experiments prove that baicalein can inhibit the PD-1/PD-L1 signal pathway in the form of molecular glue, can inhibit lung cancer development by downregulating PDL1 expression in autophagy, and can be applied as a PD-1/PD-L1 signal pathway inhibitor.

Description

PD-1/PD-L1 small molecule inhibitor and application thereof

Technical Field

The invention belongs to the technical field of medicines, relates to a PD-1/PD-L1 small molecule inhibitor, and particularly relates to a medicament for inhibiting a PD-1/PD-L1 signal path based on a molecular glue form, which inhibits lung cancer development by down regulating PDL1 expression in autophagy.

Background

Along with the deterioration of human living environment, the incidence rate of cancers is rising year by year worldwide, while lung cancer is the most common malignant tumor, and the incidence rate and the mortality rate of the lung cancer are in the forefront of all malignant tumors, and particularly in China, the lung cancer has great threat to the health and life of people in the whole society.

Non SMALL CELL Long Cancer (NSCLC) accounts for about 80-85% of total lung cancer, and has rapid disease development, rapid metastasis and high recurrence rate. At present, the treatment of the non-small cell lung cancer has become a difficult point of clinical treatment, and the method for detecting the new and more useful treatment of the non-small cell lung cancer has very important clinical significance for improving the curative effect, prolonging the survival time and improving the prognosis.

Tumor cells can break through host defense mechanisms to escape immunologically, and thus grow continuously. Cancer Immunotherapy (ICB) has been the focus of cancer research. ICB therapy against programmed cell death 1 (PD-1) and its ligand programmed death ligand 1 (PD-L1) has shown significant clinical benefit in different cancers. Clinical studies prove that over-expression of the PD-L1 gene can enable NSCLC to generate immune escape, and inhibition of the PD-L1 gene can provide assistance for NSCLC treatment. PD-1/PD-L1 can negatively regulate and control immune response by inhibiting T cell activation and proliferation, and plays an important role in regulating immune escape of tumors and the like. With intensive research into the mechanism of tumor immune tolerance and escape, PD-L1 was found to be involved in activation of CD4 ⁺ T cells, release of cytokines, and cytotoxic effects of CD8 ⁺ CTLs, mainly through CD28 binding to T cells. PD-1 binding to PD-L1 can prevent immune cells from attacking cancer cells, thereby evading detection of the immune system, resulting in further spread and progression of cancer.

PD-L1 is a type I transmembrane protein, containing two extracellular immunoglobulin (Ig) like domains and a short cytoplasmic domain, whose expression protects cancer cells from T-cell mediated immune surveillance. Early scholars thought that an important mechanism of action of PD-L1 in tumor immune escape is that the interaction between PD-L1 on tumor cells and PD-1 receptors on T cells results in inhibition of tumor killing activity and inflammatory cytokine secretion by T cells. However, in recent years, intensive researches have found that a great number of T lymphocytes are also infiltrated in the microenvironment of tumor tissues, and most of the T lymphocytes can express PD-L1, so that the PD-1/PD-L1 signals also influence the differentiation and the functions of the T cells, and the PD-1/PD-L1 has irreplaceable effects in the processes of inhibiting the generation of tumor immune response, the generation of tumor immune escape and the like. Inhibiting the combination of PD-1/PD-L1 can prevent the immune escape of cancer, relieve the inhibition of T/B cells to a certain extent and realize the immunotherapy.

Currently, monoclonal antibody assays for clinical PD-1/PD-L show high patient response rates, especially for melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, etc., for which treatment of the above indications has been approved by the FDA. The two natural ligands of the PD-1/PD-L pathway are PD-L1 and PD-L2, wherein the PD-L1 has wider expression range, is expressed in partial immune cells, tumor cells and few normal cells of the organism, and the PD-L1 is easier to combine with the PD-1, so that the PD-L1 becomes a main ligand of the PD-1, and the research of the PD-L1 also becomes a research target point of the PD-1/PD-L pathway inhibitor. Studies show that the deletion or decline of PD-L1 molecular expression of tumors can promote T cell activation, thereby improving the immune activity of T cells and inhibiting the metastasis of tumors.

For 5 months 2016, TECENTRIQ was FDA approved as a second line drug, indicated by metastatic/recurrent bladder epithelial cancer. At present, PD-L1 is taken as a target point, but a lot of monoclonal antibody medicines which are not marketed in the clinical test stage still exist. The efficacy of these monoclonal antibody drugs varies from person to person, but the following problems remain: 1. benefit limitation, patients who truly benefit from lung cancer are less than 20%; 2. there is a delay effect, and the effect of immunotherapy includes an immune activation process, which requires a certain time to establish an immune response, and then is converted into a long-term clinical effect; 3. part of patients have the condition of burst progress after medication, and tumors are accelerated to increase instead of shrinking after using PD-1/PD-L1 monoclonal antibody medicaments, and inflammatory storm and autoimmune diseases are caused.

At present, although the PD-1/PD-L1 blocking method has remarkable effect in clinical treatment of cancer patients, only a few patients can show lasting curative effect in the PD-1/PD-L1 blocking treatment, and the problems of inherent drug resistance and the like in the PD-1/PD-L1 treatment and caused inflammatory storm and autoimmune diseases still remain a relatively troublesome challenge in the current clinical and basic research. Especially for lung cancer patients, the clinical significance, biological functions and action mechanism of PD-L1 abnormal expression are urgently discussed.

In-depth research on the regulation process of PDL1 expression plays a crucial role in tumor immune escape. Although monoclonal antibody drugs against PD-L1 are a hot topic, little research has been reported on small molecule inhibitors, and no small molecule inhibitors have yet entered the clinic. The search for and development of new PD-1/PD-L1 small molecule inhibitors is therefore a challenge for researchers.

Unlike PD-1/PD-L1 monoclonal antibody medicine, which has the disadvantages of complex production process, high production cost, immunogenicity, relatively poor stability, inconvenient administration and the like, the PD-1/PD-L1 small molecule inhibitor has the advantages of targeting intracellular targets, lower production cost, higher stability, better tumor penetrability, more convenient oral administration, immunogenicity elimination and the like, so that the development of the novel PD-1/PD-L1 small molecule inhibitor is very significant.

Molecular glue refers to a class of small molecule compounds capable of mediating protein-protein interactions, and if one protein molecule is ubiquitin ligase, the molecular glue can cause ubiquitin modification of another protein and degradation through a proteasome pathway. Unlike small molecule inhibitors in the traditional sense, molecular glue is a new strategy capable of inactivating targets which are difficult to treat by traditional pharmacological methods, can avoid the limitations of the traditional inhibitors, and can accelerate the natural degradation of certain proteins on or off cells just like a molecular scalpel. Proteins are critical to many biological processes, but some proteins also help to spread diseases such as cancers, evade immune regulation, such as PD-L1, promote degradation or inactivation of such proteins, and prevent and inhibit the occurrence and development of related diseases.

Disclosure of Invention

The invention aims to provide a PD-1/PD-L1 small molecule inhibitor, which can inhibit a PD-1/PD-L1 signal path through a molecular glue form and inhibit lung cancer development through down-regulating PDL1 expression in autophagy.

Natural products have been a valuable source for the discovery of new drugs, and traditional chinese medicine from plants, animals and microorganisms have contributed significantly to human health. According to the research of the invention, the natural small molecular baicalein can regulate and control the expression of PDL1 in lung cancer tissues, and plays a role of molecular glue in the process of regulating and controlling PDL1 through a series of molecular biotechnology. The discovery not only provides a new mechanism for the expression regulation of PDL1, but also provides a thought for the development of new PDL1 regulation small molecules, and has great potential application in clinic.

Therefore, the invention firstly provides a PD-1/PD-L1 small molecule inhibitor, wherein the small molecule inhibitor comprises an active ingredient baicalein or a structural analogue thereof and is used for inhibiting the combination of PD-1 and PD-L1.

Baicalein is a flavonoid compound with a definite chemical structure, and is extracted from the dry root of the traditional Chinese medicinal plant baical skullcap root. As an active ingredient of the scutellaria baicalensis extract, baicalein exhibits various pharmacological activities such as antioxidant effect, anti-inflammatory effect, anticancer effect and antiallergic effect, but has few reports on its effect in tumor immunity.

The research of the invention discovers that natural small molecular baicalein can regulate the level of PDL1 in tumor cells, thereby inhibiting the occurrence and development of lung cancer. The invention researches the anti-tumor and immunoregulation effects of baicalein on lung cancer from outside and inside, discovers that the baicalein can reduce the expression of PD-L1, and reveals the novel inhibition effect of the baicalein on PDL1 in lung cancer tissues, thereby effectively inhibiting the occurrence and development of non-small cell lung cancer. Baicalein can reduce PD-L1 level in tumor tissue, enhance anti-tumor immunity of CD8 ⁺ T cells, and achieve similar drug effect to PDL1 antibody.

Based on the above, the invention further provides application of the small molecule inhibitor in preparing PD-1/PD-L1 signal path inhibitor.

In the deep research of the mechanism, the invention discovers that baicalein can reduce the PDL1 level in tumor tissues, but has no great influence on the PDL1 transcription process. This suggests that the manner in which baicalein regulates PD-L1 protein may be independent of the regulation of transcription levels, but that PD-L1 protein levels are reduced by affecting protein stability.

Therefore, the invention also provides further application of the small molecule inhibitor, wherein the small molecule inhibitor is used as a molecular glue medicament, and the PD-1/PD-L1 signal path is inhibited in the form of molecular glue.

Currently common protein degradation pathways include the ubiquitin-proteasome pathway and the autophagy-lysosomal pathway. After treatment with the proteasome inhibitor MG132 and the autophagy inhibitor CQ, the present invention found that only CQ significantly inhibited the decrease in PD-L1 protein levels caused by baicalein, with no significant change in MG132 dry prognosis.

Therefore, more specifically, the small molecule inhibitor provided by the invention is used as a molecular glue medicine to inhibit the development of lung cancer by down-regulating PD-L1 expression in autophagy to inhibit the PD-1/PD-L1 signaling pathway.

The autophagy inhibitor CQ can inhibit the increase of autophagy flux caused by baicalein, and the autophagy activator AZD-8055 can obviously enhance autophagy flux in combination with the baicalein. The corresponding data indicate that baicalein promotes PDL1 degradation mainly through the autophagy-lysosomal pathway. In the course of baicalein promoting the degradation of PDL1 via the autophagy-lysosomal pathway, the present invention also mimics the discovery that baicalein can bind to PDL1, thereby introducing PDL1 into the autophagy-lysosomal pathway. In fact, small molecules, when promoting protein degradation, can intervene in this process by acting as molecular glues.

Based on the facts, the invention verifies that baicalein plays a role of molecular glue in the process of promoting the degradation of PDL1, and the corresponding change of the expression of autophagy key protein LC3 is verified in the process of introducing PDL1 into autophagy flow, so that the baicalein can effectively reduce the expression of tumor PDL1 and play an anti-tumor role.

Furthermore, the invention also provides a method for reducing the expression level of PD-L1 in tumor cells, which is to add a proper amount of active ingredient baicalein or a structural analogue thereof into a culture medium in which the tumor cells are cultured.

According to the invention, through a series of in-vitro experiments, baicalein can reduce the expression of PDL1 in lung cancer tissues, and avoid immune escape, so that occurrence and development of lung cancer are inhibited. In the research of the corresponding mechanism, the baicalein can play a role of molecular glue, promote the mutual combination of PDL1 and autophagy key proteins, further accelerate the degradation of PDL1 and realize the reduction of PDL1 expression.

Drawings

FIG. 1 shows that baicalein acts like a PD-L1 antibody in vivo to inhibit tumor growth.

FIG. 2 shows that baicalein enhances anti-tumor immunity in vivo by reducing PD-L1.

FIG. 3 is a graph showing that baicalein is effective in reducing tumor cell PD-L1 protein levels.

FIG. 4 is the promotion of PD-L1 protein degradation by baicalein via the autophagy pathway.

Detailed Description

The following describes in further detail the embodiments of the present invention with reference to the drawings and examples. The following examples are presented only to more clearly illustrate the technical aspects of the present invention so that those skilled in the art can better understand and utilize the present invention without limiting the scope of the present invention.

The experimental methods, production processes, apparatuses and devices involved in the embodiments of the present invention, the names and abbreviations thereof all belong to the names conventional in the art, and are well-known and clear in the relevant fields of use, and those skilled in the art can understand the conventional process steps according to the names and apply the corresponding devices, and implement the methods according to the conventional conditions or the conditions suggested by the manufacturer.

The various materials or reagents used in the examples of the present invention are not particularly limited in source, and are conventional products commercially available. The preparation may also be carried out according to conventional methods known to the person skilled in the art.

Example 1: effect of baicalein on tumor growth in lung cancer cell engrafting tumor mice.

The influence of baicalein on the growth of tumors in lung cancer cell transplantation tumor mice is studied in the embodiment so as to verify whether the mice have corresponding curative effects of PDL1 monoclonal antibodies.

Lewis lung cancer cells (LLC) were injected subcutaneously into C57BL/6 mice, and then baicalein or anti-PD-L1 mab was administered for 15 days, respectively, and the experimental flow chart of the transplanted tumor model is shown in FIG. 1 (A).

As can be seen from the tumor tissue images 15 days after the baicalein and anti-PD-L1 mab treatment of fig. 1 (B), the tumors of the mice in the baicalein-treated group and the anti-PD-L1 mab-treated group were significantly reduced compared to the control group.

Furthermore, as shown in the tumor tissue growth curves of mice in each group in FIG. 1 (C), the tumor growth rate of tumor-bearing mice was significantly reduced after 15 days of treatment with baicalein and anti-PD-L1 monoclonal antibody, respectively, compared with the control group.

Further analysis of tumor volume in mice 15 days after treatment revealed that tumor volumes in baicalein-treated and anti-PD-L1 mab-treated mice were significantly reduced after 15 days of treatment compared to control group, as shown in fig. 1 (D).

To assess the safety of the drug, the mice weights were measured every other day, as shown in fig. 1 (E), with no significant decrease in weight for each group of mice during treatment.

The results prove that the baicalein plays an anti-tumor role similar to that of the PD-L1 antibody in vivo, and the anti-tumor growth effect of the baicalein is equivalent to that of anti-PD-L1 monoclonal antibody treatment.

Example 2: baicalein enhances anti-tumor immunity in vivo by reducing PD-L1.

In order to explore the influence of baicalein on the PD-L1 level and immunity of tumors, the PD-L1 level and corresponding effector molecules in tumor tissues are further explored.

Immunohistochemical experiments detect the level of PD-L1 protein in the tumor tissues of mice in each group, and as shown in FIG. 2 (A), compared with the control group, the obvious reduction of PD-L1 protein in the tumor tissues of mice in the baicalein treatment group and the anti-PD-L1 monoclonal antibody treatment group can be seen.

Meanwhile, the number and the duty ratio of PD-L1 positive tumor cells in tumor tissues of tumor-bearing mice are detected and analyzed by adopting a flow cytometry, and the results are shown in (B) and (C) in FIG. 2 respectively. Compared with the control group, the absolute value and the percentage of PD-L1 MFI and PD-L1 positive tumor cells in the tumor tissue of the baicalein treatment group are obviously reduced. The experimental results show that the baicalein can reduce the PD-L1 level of tumor cells.

Numerous studies have previously shown that after binding of PD-L1 on tumor cells to PD-1 on T cells, tumor immune escape is promoted and tumor cell growth is promoted. Based on the above, the invention further verifies whether the reduction of the PD-L1 level by baicalein can affect the anti-tumor immunity.

As shown in the immunohistochemical experimental results of (D) in FIG. 2, the levels of Granzyme B and Perforin in the tumor tissue of the mice in the baicalein-treated group were significantly increased as compared to the control group.

It is well known that Granzyme B and Perforin are effector molecules secreted by CD8 ⁺ T cells and can directly kill tumor cells. The results comprehensively show that the baicalein can enhance the anti-tumor immunity of CD8 ⁺ T cells by reducing the PD-L1 level in tumor tissues in vivo and exert the effect similar to the anti-PD-L1 monoclonal antibody, thereby inhibiting the tumor growth.

Example 3: baicalein is effective in reducing tumor cell PD-L1 protein level.

In order to deeply investigate the mechanism of baicalein for regulating the level of PD-L1 in tumor cells, the influence of baicalein on the level of PD-L1 protein in tumor cells is studied by adopting a WB method.

As shown in fig. 3 (a), the WB experiment examined PD-L1 protein levels after intervention of lung cancer cells for 48H with different concentrations of baicalein in different tumor cell lines (LLC/a 549/H1299), and the results indicated that baicalein was able to decrease PD-L1 protein levels in tumor cells in a dose-dependent manner.

Similarly, after 48h of baicalein intervention, the immunofluorescence assay results of IF detection of PD-L1 protein levels also showed that baicalein reduced tumor cell PD-L1 protein expression in a dose-dependent manner (FIG. 3B).

However, the corresponding q-PCR experimental results in FIG. 3 (C) show that baicalein dry prognosis does not affect the mRNA expression of tumor cell PD-L1. This suggests that the manner in which baicalein regulates PD-L1 protein may be independent of the regulation of transcription levels, but rather by affecting the manner in which protein stability is affected, thereby reducing PD-L1 protein levels.

As shown in FIG. 3 (D), total protein lysates were extracted from A549 cells, and incubated with biotin and bio-baicalein, respectively, for IP experiments, and the experimental results showed that baicalein could bind to PD-L1 protein.

Further molecular docking analysis was used to investigate the interaction between baicalein and PD-L1 protein, and the results are shown in FIG. 3 (E).

Example 4: baicalein promotes PD-L1 degradation through the autophagy pathway.

Baicalein does not affect the transcript level of lung cancer cells PD-L1, indicating that the regulation of PD-L1 by baicalein may affect the stability of its protein by relying on protein degradation pathways.

Common protein degradation pathways include the ubiquitin-proteasome pathway and the autophagy-lysosomal pathway. Thus, this example determines whether the post-translational pathway correlates with decreased expression of PD-L1 in lung cancer cells following baicalein intervention by using the proteasome inhibitor MG132 and the autophagy inhibitor CQ.

After intervention of a549, H1299 and LLC cells with MG132, CQ and baicalein, respectively, WB detected PD-L1 protein levels. As shown in fig. 4 (a), in a549, H1299 and LLC cells, only CQ significantly inhibited the decrease in PD-L1 protein level caused by baicalein, and MG132 dry prognosis was not significantly changed. These results indicate that baicalein may promote the degradation of PD-L1 by the autophagy-lysosomal pathway.

Next, WB detects LC3B-I/II protein levels after baicalein intervenes in a549, H1299 and LLC cells, further discussing the effect of baicalein on tumor cell autophagy levels. As shown in FIG. 4 (B), baicalein intervention significantly increased LC3-I to LC3-II conversion in A549, H1299 and LLC cells.

Further, after transfecting H1299 cells for 24 hours by adopting RFP-GFP-LC3 double-labeled probes, the H1299 cells are treated by baicalein, CQ and AZD8055 for 24 hours, and the influence of the baicalein on autophagy flux of tumor cells is studied.

The immunofluorescence picture of fig. 4 (C) shows a representative image of the fluorescent LC3 spot. The results showed that after baicalein intervention, H1299 cell autophagy was significantly increased, which was manifested by reduced GFP-LC3 spot formation and total GFP fluorescence signal, showing a red appearance in the pooled images.

Meanwhile, the autophagy inhibitor CQ can inhibit the increase of autophagy flux caused by baicalein, and the autophagy activator AZD-8055 and baicalein can also obviously enhance autophagy flux. These results all indicate that baicalein promotes PD-L1 protein degradation by enhancing autophagy.

The above embodiments of the invention are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Various changes, modifications, substitutions and alterations may be made by those skilled in the art without departing from the principles and spirit of the invention, and it is intended that the invention encompass all such changes, modifications and alterations as fall within the scope of the invention.

Claims (5)

1. A PD-1/PD-L1 small molecule inhibitor comprises an active ingredient baicalein or a structural analogue thereof, and is used for inhibiting the combination of PD-1 and PD-L1.

2. Use of the small molecule inhibitor of claim 1 for the preparation of a PD-1/PD-L1 signaling pathway inhibitor.

3. The use according to claim 2, characterized in that the small molecule inhibitor is used as a molecular glue drug to inhibit the PD-1/PD-L1 signaling pathway in the form of molecular glue.

4. The use according to claim 2, wherein the small molecule inhibitor is used as a molecular glue drug to inhibit the development of lung cancer by down-regulating PD-L1 expression in autophagy to inhibit the PD-1/PD-L1 signaling pathway.

5. A method for reducing PD-L1 expression in tumor cells comprises adding baicalein or its structural analogue as active ingredient into culture medium containing tumor cells.