阅读说明：本技术 丁香酸葡萄糖苷在制备抑制pd1和pd-l1蛋白之间相互作用药物中的应用 (Application of syringic acid glucoside in preparation of medicines for inhibiting interaction between PD1 and PD-L1 protein ) 是由 董子钢 刘康栋 李侎泫 顾廷轩 田雪利 王园园 于 2020-05-07 设计创作，主要内容包括：本发明公开一种丁香酸葡萄糖苷在制备抑制PD1和PD-L1蛋白之间相互作用药物中的应用,属于医药领域。本发明经研究发现：丁香酸葡萄糖苷可以作为PD1/PD-L1蛋白相互作用的小分子抑制剂。PD1/PD-L1信号通路与免疫应答、肿瘤免疫逃逸密切相关,抑制PD1/PD-L1信号通路的异常激活能够起到预防及治疗肿瘤的作用。本发明的丁香酸葡萄糖苷能够抑制PD1/PD-L1蛋白质之间相互作用。(The invention discloses an application of syringic acid glucoside in preparation of a medicine for inhibiting interaction between PD1 and PD-L1 protein, and belongs to the field of medicines. The research of the invention finds that: syringic acid glucoside can be used as a small molecule inhibitor of PD1/PD-L1 protein interaction. The PD1/PD-L1 signal channel is closely related to immune response and tumor immune escape, and the effect of preventing and treating tumors can be achieved by inhibiting abnormal activation of the PD1/PD-L1 signal channel. The syringic acid glucoside disclosed by the invention can inhibit the interaction between PD1/PD-L1 proteins.)

1. The application of syringic acid glucoside in the preparation of small molecule immunotherapy medicaments is characterized in that the syringic acid glucoside is used as an inhibitor of the interaction between PD1 and PD-L1 protein.

2. The use of syringic acid glucoside in the preparation of small molecule immunotherapeutic agents as claimed in claim 1, wherein the concentration of PD1 is 10 μ g/ml, the concentration of PD-L1 is 10 μ g/ml, and the concentration of syringic acid glucoside is 2-80 μ M.

3. Application of syringic acid glucoside in preparation of lung cancer immunotherapy medicament is provided.

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of syringic acid glucoside in preparation of a medicine for inhibiting interaction between PD1 and PD-L1 protein.

Background

Novel cancer immunotherapy is the most promising cancer treatment strategy, mainly involving chimeric antigen receptor T cells, bispecific antibodies and immune checkpoint inhibitors. The programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) axis is an important immune checkpoint signal pathway, and can reduce the amplitude of inflammatory reaction and maintain the immune homeostasis of the body. These pathways involve immune homeostasis or employ features that allow tumor cells to actively evade immune cell elimination. They are inhibitors of T cell proliferation and function. It has been shown that targeting immune checkpoint inhibitors, overcoming the intrinsic resistance of immune surveillance by enhancing the anti-tumor immune response, by blocking the interaction of PD-1 with its ligands, stimulates the T cell response. This also leads to improved results and increased patient survival.

Recent experiments have demonstrated that clinically successful cancer immunotherapy includes the regulatory approval of the T cell checkpoint inhibitory antibodies ipilimumab, pembrolizumab and nivolumab. Iplilimumab is a human IgG1k monoclonal antibody that binds to the extracellular domain of human CTLA-4 with high affinity and is an inhibitor of the complex function. Ipilimumab causes activation and proliferation of T cells, infiltration of lymphocytes, and death of tumor cells. The enhancement of effector T cell function, suppression of CD4+ Treg cells and CD8+ suppressor cells, is critical for ipilimumab to exert its therapeutic effect. Pembrolizumab and nivolumab are humanized monoclonal antibodies that prevent the participation of ligands, thereby interfering with T cell signaling and cell death. All of these monoclonal antibodies showed significant clinical benefit to melanoma as single agents and were continuously tested in clinical trials as single agents and combinations. These successful immunotumor drugs take advantage of the unique immune mechanisms associated with cancer growth and treatment, from early tumor-associated antigen reaction to relief of T cell activation and tumor immunosuppression.

Compared with recombinant protein drug design, the small molecule compound has the following obvious advantages: 1) the clinical application and development of the method have detailed knowledge and historical precedent, and the method has higher feasibility; 2) oral bioavailability; 3) exposure to the tumor microenvironment or crossing physiological barriers (such as the blood brain barrier); 4) obtaining intracellular disease targets which are difficult to control by protein therapeutic drugs; and 5) diverse and well-understood formulation and dosage selection to alleviate pharmacokinetic and/or pharmacodynamic challenges and to expose titrated drugs. Another compelling advantage of small molecule drugs is that these types of drugs are more readily available to patients than biological immunotherapy. The cost of small molecule drugs is generally lower due to the drug itself, the reduced cost of delivering the tablet compared to infusion (or injection), and the simpler supply chain, not requiring refrigeration. In order for immunotherapy to deliver its full potential in modifying cancer treatment, the cost must be reduced to allow treatment to be available to all persons who might benefit. Even though cost is not an obstacle to acquisition, the convenience of oral drugs relative to infusion is a strong difference from the currently developing treatments of immune tumors.

Disclosure of Invention

The invention aims to overcome the defects of a monoclonal antibody and provides application of a small molecular compound syringic acid glucoside in preparing a medicament for inhibiting interaction between PD1 and PD-L1 protein. Syringic acid glucoside is a small molecular compound, and the molecular formula is as follows: c₁₅H₂₀O₁₀Molecular weight: 360.3, CAS number 33228-65-8.

The invention provides application of a small molecular compound syringic acid glucoside in preparation of a medicine for inhibiting interaction between PD1/PD-L1 proteins.

Specifically, the invention finds the application of syringic acid glucoside in preparing medicines for inhibiting a PD1/PD-L1 signal channel, namely the syringic acid glucoside has an inhibiting effect on a PD1/PD-L1 signal channel.

Furthermore, the concentration of PD1 is 10 mug/ml, the concentration of PD-L1 is 10 mug/ml, and the syringic acid glucoside can inhibit the in vitro combination of PD1 and PD-L1 and the in vitro interaction between PD1/PD-L1 protein when the concentration is 2-80 muM.

The invention discovers that: the application of syringic acid glucoside in preparing the medicament for inhibiting the PD1/PD-L1 signal channel is disclosed, namely the inhibition effect of syringic acid glucoside on the PD1/PD-L1 signal channel is found, and the appropriate concentration of the inhibition effect of syringic acid glucoside on the signal channel is as follows: 2-80 mu M.

The application discovers the capacity of syringic acid glucoside to bind H1975 cell lysate (rich in PD-L1) and pure proteins of Human PD 1(Cat: 10377-H03H, Human, Recombinant, Sino Biological) and Human PD-L1(Cat: 10084-H02H, Human, Recombinant, Sino Biological) in vitro.

The research of the invention finds that: syringic acid glucoside can be used as an inhibitor of PD1/PD-L1 protein interaction. The PD1/PD-L1 signal channel is closely related to immune response and tumor immune escape, and the effect of preventing and treating tumors can be achieved by inhibiting abnormal activation of the PD1/PD-L1 signal channel. The syringic acid glucoside disclosed by the invention can inhibit the interaction between PD1/PD-L1 proteins.

Drawings

FIG. 1 is a graph that tests the ability of syringic acid glucoside to bind human PD1 and human PD-L1 in vitro at different concentration gradients; wherein the syringic acid glucoside can bind to human PD-1 and human PD-L1, and has KD values of 5.631 x 10^-5 M and 3.06 x 10^-5M。

FIG. 2 is the ability of syringic acid glucoside to bind to H1975 cell lysate (enriched in PD-L1) and to human PD1 and PD-L1 pure proteins in vitro.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.

Application test

Materials and methods

Reagent

Glucrosingic acid (syringic acid glucoside) was purchased from Chem Faces and was 98% pure.

Instruments and equipment:

haier medical low-temperature storage box (Qingdao haier special electric appliance limited); enzyme linked immunosorbent assay (thermo corporation): biacore T200 (GE Healthcare Life Sciences); electronic balances (mertler-toledo instruments shanghai ltd); cell culture chambers (thermo corporation); thermo superclean bench: amino Coupling Kit (Amine Coupling Kit, GE Healthcare Life Sciences).

Syringic acid glucoside ability to bind human PD1 and PD-L1 in vitro

Human recombinant PD1 protein (histone tag), human recombinant PD-L1 protein (histone tag), was purchased from beijing yi qian shenzhou science co. The specific experimental procedure was as follows, the protein was first diluted to 10. mu.g/ml with 10mM acetate buffer (pH 4.5). Human recombinant PD1 protein and human recombinant PD-L1 protein were cross-linked on Series S sensor chip CM5 chip by amino coupling with the aid of Biacore T200 instrument, respectively, at a coupling amount of 5000 units. After the crosslinking of the recombinant protein is completed, the in vitro physical binding capacity of the compound syringic acid glucoside to the human recombinant PD1 protein (histone tag) and the human recombinant PD-L1 protein (histone tag) is detected through kinetic analysis. The syringic acid glucoside solution with final concentration of 2 μ M,10 μ M,20 μ M,40 μ M and 80 μ M respectively reacts with PD1 protein and PD-L1 protein channel on CM5 chip at flow rate of 30ul/min, and the reaction process is shown in FIG. 1. Calculating the in vitro binding capacity, K, of syringic acid glucoside, human recombinant PD1 protein and human recombinant PD-L1 protein by using Biacore analysis software_DThe values are shown in Table 1. As can be seen from Table 1 and FIG. 1, syringic acid glucoside can be clearly physically combined with human PD-1 and PD-L1 recombinant proteins in vitro.

TABLE 1

In table 1: hPD-1 represents human recombinant PD1 protein, hPD-L1 represents human recombinant PD-L1 protein.

Ability of syringic acid glucoside to bind to H1975 cell lysate and human PD1 and PD-L1 pure proteins in vitro

The specific experimental procedure is as follows, and the H1975 lung cancer cell line uses RPMI-1640 medium ((Hyclone, USA)) containing 10% fetal bovine serum (BI Co.). H1975 cells were lysed from the dish using 0.25% trypsin, centrifuged at 1000rpm for 3 minutes, and the medium was discarded. Whole cell extracts were prepared by direct lysis with 1 Xcell lysis buffer (Cat # R0020, Solarbio, Beijing, China) and protease inhibitors. All protein samples were denatured by heating at 95 ℃ for 5 minutes in a 5 x SDS buffer and then analyzed by protein electrophoresis by SDS-PAGE. Protein loading protein concentration was determined using a BCA quantification kit (Cat # PC0020, Solarbio, Beijing, China).

H1975 cell lysate (3.33 mg/mL, 20. mu.L), human PD-1 recombinant protein (0.25mg/mL, 2. mu.L), PD-L1 recombinant protein (0.25mg/mL, 2. mu.L), and syringic acid glucoside-Sepharose 4B mixture (the amount of Sepharose 4B in the mixture is 0.3g, and the amount of syringic acid glucoside is 2 mg) were added to the corresponding wells, respectively, and the electrophoresis voltage was controlled at 80V for 100 minutes. Then wet-transfer the film for 90 minutes. Blocking with 5% skim milk powder in 1 XPBS-T (phosphate buffered saline with 0.05% Tween-20) for 1 hour, followed by overnight incubation with anti-PD 1 or PD-L1 antibody. On day 2, the cells were washed 3 times in 1 XPBS-T buffer for 15 minutes each, and then incubated with horseradish peroxidase-labeled secondary antibody against the antibody. Specific protein bands were visualized using Enhanced Chemiluminescence (ECL) detection reagents and Amersham Imager 600 (GE Healthcare life Science, pittsburgh, PA, USA). Quantification was performed using Image J free software (NIH, Version 1.47, RRID: SCR-003070), and the results are shown in FIG. 2. From FIG. 2, it can be concluded that syringic acid glucoside can be combined with both hPD1 and hPD-L1.

In summary, it can be seen that: according to the invention, the syringic acid glucoside is combined with PD1 and PD-L1 through research, so that the in vitro interaction between PD1/PD-L1 protein is inhibited.