CN112370444A

CN112370444A - PD-1/PD-L1 inhibitor and application thereof

Info

Publication number: CN112370444A
Application number: CN202011283913.0A
Authority: CN
Inventors: 陈鲲; 蔡银怡; 樊静; 李文甫; 蔺佳雨; 许洁虹; 杜俊熹; 吕鹏程
Original assignee: Guangzhou University
Current assignee: Guangzhou University
Priority date: 2020-11-16
Filing date: 2020-11-16
Publication date: 2021-02-19
Anticipated expiration: 2040-11-16
Also published as: CN112370444B

Abstract

The invention relates to the field of molecular immunology, and particularly discloses a PD-1/PD-L1 inhibitor and application thereof. The PD-1/PD-L1 inhibitor of the invention comprises S-cyanomethyl-L-cysteine, by liberating H₂S promotes the activation of lymphocytes, down-regulates PD-1 gene expression and down-regulates the expression of liver cancer cell PD-L1 gene, thereby inhibiting PD-1/PD-L1 pathway, blocking cancer cell immune escape reaction, enhancing the immune response capability of lymphocytes to HepG2 cells, and enhancing the liver immune response capability of lymphocytes to liver cellsThe cancer cell inhibiting effect also enhances the immune function of mice, and can also effectively enhance the cancer inhibiting effect of quinoline derivatives of tumor targeting drug N-isostere tectoridin.

Description

PD-1/PD-L1 inhibitor and application thereof

Technical Field

The invention relates to the field of molecular immunology, in particular to a PD-1/PD-L1 inhibitor and application thereof.

Background

Primary liver cancer (PHC) is a malignant tumor caused by hepatocytes or intrahepatic bile duct epithelial cells, and the death rate of liver cancer in 2012 of our country occupies the 3 rd position of the malignant tumor, so how to effectively treat PHC has very important clinical significance. The current treatment methods of PHC can be divided into surgical treatment and non-surgical treatment, wherein the surgical treatment comprises liver transplantation and liver resection, and the non-surgical treatment methods comprise hepatic artery chemoembolization, radiofrequency ablation, radiotherapy, systemic chemical drug therapy, biological therapy, traditional Chinese medicine therapy and the like. The molecular targeted therapy has attracted attention to the strong targeting property and good curative effect, but the molecular targeted drug finally has drug resistance after acting for a period of time, so a new therapeutic strategy is to be further developed.

With the rapid development of molecular biology and molecular immunology, the immunotherapy of tumors gradually becomes a clinical research hotspot, and has the functions of stimulating specific immunity, enhancing the immune rejection of organisms to tumors, enhancing the immune rejection capability of organisms to tumors, inhibiting and killing tumors, and reducing the tumor recurrence and metastasis capability of organisms. However, multiple studies show that part of molecular targeted drugs have important immunoregulatory properties, which can affect various stages of immune effects, so that the combined action of immunotherapy and molecular targeted therapy can exert stronger anti-tumor effect. Immunotherapy is mainly divided into cell immunotherapy, cytokines, tumor vaccines, immune checkpoint inhibitors and other therapeutic strategies. Wherein the immune checkpoint inhibitor belongs to indirect immunotherapy and inhibits liver cancer cells by blocking certain specific pathways. Researches show that the expression of PD-1/PD-L1 serving as an immune checkpoint inhibitor target is increased in a tumor microenvironment, the activation of the signal channel can inhibit the activation and proliferation of T cells and the secretion of cytokines such as IL-10 and INF-gamma, and the tumor cells activate the inhibition channel of PD-1/PD-L1 by up-regulating the expression of PD-L1 molecules, inhibit the activation of the T cells and form a microenvironment suitable for the growth of the tumor cells. Immune checkpoint inhibitors can inhibit this pathway, leaving T cells uninhibited for activation, resulting in killing of tumor cells. At present, the immune checkpoint inhibitor has better curative effect in clinical tests of malignant melanoma, non-small cell lung cancer, renal cell carcinoma, bladder cancer, lymphoma and the like.

Endogenous gas molecule hydrogen sulfide (H)₂S) is known as three major gas signal molecules together with Nitric Oxide (NO), carbon monoxide (CO). Research has proved that the gaseous signal molecule hydrogen sulfide has anticancer effect. Liu' an Wen et al discloses "research progress of action of gas signal molecule hydrogen sulfide in tumorigenesis development and treatment", and the research proposes that exogenous sodium hydrosulfide generates hydrogen sulfide to activate protein kinase to activate proliferation cycle of cells, thereby inducing cell proliferation. Caluyem discloses the research on the anti-cancer effect and molecular mechanism of a hydrogen sulfide donor S-propargyl cysteine on gastric cancer cells SGC-7901, breast cancer cells MDA-MB231 and a tumor nude mouse model thereof, and experiments show that the hydrogen sulfide donor S-propargyl cysteine (SPRC) can have the anti-cancer effect on gastric cancer and breast cancer by inducing the protein activity of target protein CSE and the anti-cancer effect of hydrogen sulfide. At present, the PD-1/PD-L1 inhibitor aiming at the clinical test of liver cancer has relatively few researches and applications, so that the improvement space exists.

Disclosure of Invention

It is a first object of the present invention to overcome the deficiencies of the prior art described above and to provide a PD-1/PD-L1 inhibitor by releasing H₂S promotes lymphocyte activation, down-regulates PD-1 gene expression, down-regulates the expression of liver cancer cell PD-L1 gene, thereby inhibiting PD-1/PD-L1 access, blocking cancer cell immune escape reaction,so as to enhance the immune response capability of lymphocytes to HepG2 cells, enhance the inhibition effect of the lymphocytes to liver cancer cells, effectively enhance the immune function of mice, and effectively enhance the cancer inhibition effect of the quinoline derivatives of the tumor-targeted medicament N-isostere tectoridin.

In order to achieve the purpose, the invention adopts the technical scheme that:

a PD-1/PD-L1 inhibitor comprises S-cyanomethyl-L-cysteine.

A PD-1/PD-L1 inhibitor comprises quinoline derivatives of S-cyanomethyl-L-cysteine and N isostere tectoridin.

In the technical scheme of the invention, when the PD-1/PD-L1 inhibitor comprises S-cyanomethyl-L-cysteine (SCC), H can be released₂S promotes lymphocyte activation and down regulates the expression level of lymphocyte PD-1 and HepG2 cells PD-L1, and experimental examples 3.3-3.4 show that SCC can not only down regulate the expression of lymphocyte PD-1 protein, but also down regulate the expression of lymphocyte PD-1 and HepG2 cells PD-L1 mRNA, so that the combination of the secreted PD-1 ligand (PD-L1) and lymphocyte surface PD-1 is reduced, the combination of the PD-1 ligand (PD-L1) secreted by HepG2 and lymphocyte surface PD-1 is reduced, the activation of lymphocytes is enhanced, the transformation rate of lymphocytes is improved, the immune function of mice is enhanced, and the proliferation of HepG2 cells is effectively inhibited.

When the PD-1/PD-L1 inhibitor included quinoline derivatives of S-cyanomethyl-L-cysteine and N-isosteric tectoridin, HepG2 cells were significantly reduced in number, lymphocyte inhibition of HepG2 cells was increased (P <0.01), survival of HepG2 cells was decreased, and it was found from test example 4 that HepG2 was only 17% inhibited when treated with 10 μ M SCC; when the quinoline derivative of N-isostearic tectoridin is used for treatment alone, the inhibition rate of HepG2 is only 31%, but when the quinoline derivative of SCC and N-isostearic tectoridin is used in combination, the inhibition rate of HepG2 cells is 66%, the inhibition rate of HepG2 cells is remarkably improved, and the result shows that the SCC can effectively enhance the cancer inhibition effect of the quinoline derivative of the N-isostearic tectoridin serving as a tumor targeting drug.

In a preferred embodiment of the inhibitor of the present invention, the concentration of S-cyanomethyl-L-cysteine is 1 to 100. mu.M.

With the increase of the concentration of S-cyanomethyl-L-cysteine, the inhibition effect of lymphocytes on HepG2 cells is enhanced, and the conversion rate of the lymphocytes is obviously improved (P < 0.01); when the concentration of S-cyanomethyl-L-cysteine is 1 mu M, the expression level of lymphocyte PD-1 protein is obviously reduced (P < 0.01).

As a preferred embodiment of the inhibitor of the present invention, the preparation method of S-cyanomethyl-L-cysteine comprises the steps of:

s1, dissolving raw material L-cysteine in NH₄In the OH solution, stirring to obtain a mixture;

s2, dropwise adding 2-bromoacetonitrile into the mixture, stirring overnight, carrying out vacuum concentration and filtration, and recrystallizing the filtrate to obtain S-cyanomethyl-L-cysteine.

In a preferred embodiment of the inhibitor of the present invention, in the step S1, the stirring temperature is-10 to 10 ℃.

As a preferred embodiment of the inhibitor of the present invention, in the step S2, the filtrate is recrystallized by using a water-ethanol solution with a volume ratio of (1-3) to (2-4).

The second purpose of the invention is to provide an application of the inhibitor in preparing anti-liver cancer drugs or preparations.

As a preferred embodiment of the use according to the invention, the S-cyanomethyl-L-cysteine is liberated from H₂S promotes the activation of lymphocytes, down-regulates the expression of PD-1 gene, down-regulates the expression of PD-L1 gene in liver cancer cells, and inhibits the PD-1/PD-L1 pathway, thereby promoting the apoptosis of the liver cancer cells.

As a preferred embodiment of the use of the invention, the S-cyanomethyl-L-cysteine promotes apoptosis of N-isosteric tectoridin quinoline derivatives on hepatoma cells.

As a preferred embodiment of the use of the present invention, the liver cancer cells include HepG2 cells.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention provides a novel PD-1/PD-L1 inhibitor, wherein S-cyanomethyl-L-cysteine (SCC) can promote the activation of lymphocytes by releasing H2S, reduce the expression of PD-1 genes and reduce the expression of PD-L1 genes of HepG2 cells, thereby inhibiting a PD-1/PD-L1 pathway, blocking the immune escape reaction of tumor cells and enhancing the immune response capability of the lymphocytes to the HepG2 cells.

(2) The present invention provides a novel PD-1/PD-L1 inhibitor which, when used in combination with N-isosteric tectoridin quinoline derivatives, can also enhance the anti-cancer effect of N-isosteric tectoridin quinoline derivatives and inhibit the proliferation of HepG2 cells.

Drawings

FIG. 1 is a graph showing the phagocytic activity of SCC in mice in low, medium and high dose groups on macrophages in the abdominal cavity;

FIG. 2 is a graph showing the effect of different concentrations of SCC on the inhibition rate of HepG2 cells;

FIG. 3 is a schematic representation of different concentrations of SCC-activated mouse lymphocytes;

FIG. 4 is a graph showing the effect of different concentrations of SCC on lymphocyte activation rate;

FIG. 5 is a graph showing the effect of different concentrations of SCC on lymphocyte PD-1 protein expression levels;

FIG. 6 is a graph showing the effect of different concentrations of SCC on lymphocyte PD-1mRNA expression levels;

FIG. 7 is a graph showing the effect of different concentrations of SCC on the expression level of PD-L1 mRNA from HepG2 cells;

FIG. 8 is a graph showing the effect of S-cyanomethyl-L-cysteine in combination with a quinoline derivative of N-isostere tectoridin on cell survival in HepG 2.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

In the following examples, the N-isosteric tectoridin quinoline derivative (New Tectrorigenin) of the present invention was prepared as disclosed in CN108685921 b.

Among them, Wright-Giemsa dye liquor (Wright-Giemsa, available from Shanghai Biyutian Biotech Co.), EnhancedCellCountingkit-8 (available from Shanghai Biyutian Biotech Co.), RNAioso Plus kit (available from TaKaRa Co., lot: 9108), Prime Script^TMRT reagent kit Kitwithg DNA Eraser kit (from TaKaRa company, batch No.: RR 047A).

Example 1

A method for preparing S-cyanomethyl-L-cysteine (SCC) comprises the following steps:

s1, dissolving 200mg of L-cysteine with the mass concentration of 1.65mmol in 2mL of NH with the mass concentration of 2M₄Stirring in OH solution to obtain mixture at-10 deg.C for 30 min;

s2, dripping 218mg of 2-bromoacetonitrile with the mass concentration of 1.82mmol into the mixture, stirring overnight, concentrating and filtering under the condition that the vacuum degree is 0.08MPa, washing the filtrate with ethanol, and then recrystallizing with a water-ethanol solution with the volume ratio of 1:2 to obtain light yellow solid S-cyanomethyl-L-cysteine.

Example 2

s1, dissolving 200mg of L-cysteine with the mass concentration of 1.65mmol in 2mL of NH with the mass concentration of 2M₄Stirring the OH solution to obtain a mixture, wherein the stirring temperature is 10 ℃, and the stirring time is 30 min;

s2, dripping 218mg of 2-bromoacetonitrile with the mass concentration of 1.82mmol into the mixture, stirring overnight, concentrating and filtering under the condition that the vacuum degree is 0.08MPa, washing the filtrate with ethanol, and then recrystallizing with a water-ethanol solution with the volume ratio of 3:4 to obtain light yellow solid S-cyanomethyl-L-cysteine.

Example 3

s1, dissolving 200mg of L-cysteine with the mass concentration of 1.65mmol in 2mL of NH with the mass concentration of 2M₄In OH solution, stirring to obtainStirring the mixture at 0 ℃ for 30 min; s2, dripping 218mg of 2-bromoacetonitrile with the mass concentration of 1.82mmol into the mixture, stirring overnight, concentrating and filtering under the condition that the vacuum degree is 0.08MPa, washing the filtrate with ethanol, and then recrystallizing with a water-ethanol solution with the volume ratio of 2:2 to obtain light yellow solid S-cyanomethyl-L-cysteine.

Example 4

The invention provides a PD-1/PD-L1 inhibitor, which comprises S-cyanomethyl-L-cysteine or/and quinoline derivatives of S-cyanomethyl-L-cysteine and N-isosteric tectoridin.

The invention also provides an application of the inhibitor in preparing an anti-liver cancer medicine or preparation.

The S-cyanomethyl-L-cysteine is released by releasing H₂S promotes the activation of lymphocytes, down-regulates the expression of PD-1 gene, down-regulates the expression of PD-L1 gene in liver cancer cells, and inhibits the PD-1/PD-L1 pathway, thereby promoting the apoptosis of the liver cancer cells; the S-cyanomethyl-L-cysteine promotes the apoptosis of the quinoline derivative of the N-isostere tectoridin on liver cancer cells.

Test examples

1. Material preparation

1.1 isolation of Primary splenic lymphocytes

Adult healthy Balb/c mice are killed by cervical dislocation, soaked in 75% ethanol for 10s, and the spleens of the mice are cut under aseptic conditions. Mechanically separating splenocytes, grinding with PBS, filtering with 200 mesh filter screen, removing erythrocytes with erythrocyte lysate, centrifuging, washing with PBS for 2 times, centrifuging again, adding culture solution to obtain cell suspension, culturing in culture flask for 12 hr, sucking cell suspension, purifying T cells, separating to 6-well plate, adjusting cell concentration to 1 × 10⁸And L. Adult healthy Balb/c mice are killed by cervical dislocation, soaked in 75% ethanol for 10s, and the spleens of the mice are cut under aseptic conditions. Mechanically separating splenocyte, grinding with PBS, filtering with 200 mesh filter screen, removing erythrocyte with erythrocyte lysate, centrifuging, cleaning with PBS for 2 times, centrifuging again, adding culture solution to obtain cell suspension, culturing in culture flask for 12 hr, sucking cell suspension, purifying T cell, and separating to obtain6 well plates, adjusted to 5X 10 cell concentration⁹/L。

1.2 SCC treatment of lymphocytes

The solid S-cyanomethyl-L-cysteine prepared in example 3 was pulverized, SCC was dissolved in PBS, and the resulting mixture was stored at 4 ℃ and diluted with a cell culture medium to a desired concentration at the time of use. Different doses of SCC (final concentrations of 1. mu.M, 10. mu.M and 30. mu.M) were added to the six-well plate in step 1.1, and the control group was replaced with an equal amount of DMEM medium, and the plate was placed in an incubator for 48 hours, and the growth of lymphocytes at each concentration group was observed under an inverted microscope and recorded by photography. The number of cells treated with SCC at different concentrations for 48h was counted by image J software, and the proliferation rate was calculated.

Lymphocyte transformation ratio (%) × 100% (cells with moderate transformation and higher/100 lymphocytes).

1.3 Resuscitation and culture of HepG2 liver cancer cells

Taking out the frozen HepG2 cells from a liquid nitrogen tank, quickly placing the cells in a constant-temperature water bath kettle at 37 ℃, quickly shaking, transferring cell suspension in a super-clean workbench after the cells are ablated, centrifuging for 5min at 800rpm and 25 ℃, collecting cell precipitates, adding a proper amount of culture solution (using low-sugar DMEM culture solution containing 2% 100u/mL double antibody and 10% FBS), blowing, uniformly mixing, transferring to a culture bottle, and placing in an incubator for culture. The culture conditions are as follows: 5% CO₂And saturated humidity at 37 ℃. After 12h of culture, the effect of resuscitating HepG2 cells was observed using an inverted phase contrast microscope, nonadherent floating as dead cells or cell debris, adherent or differentiated as live cells.

2. Animal experiments

2.1 animal grouping and handling

12 male Balb/c mice are taken and fed with common feed, the feed is adapted for 1 week, then the male Balb/c mice are averagely divided into 4 groups which are respectively a blank control group, an SCC low-dose group, an SCC medium-dose group and an SCC high-dose group, and a mouse immunosuppressive model is established. Mice were injected subcutaneously for seven consecutive days: the same volume of physiological saline (blank control group); ② 5mg/kg (SCC low dose group); ③ 10mg/kg (in SCC dose group); (iv) 20mg/kg (SCC high dose group).

The breeding conditions of the mice are as follows: the basic feed is freely taken and eaten, the air humidity is 60 +/-5%, and the temperature is 25-27 ℃.

2.2 measurement of phagocytic function of macrophages in the peritoneal cavity

And 7d, subcutaneous injection administration is carried out for about 4-6 hours, namely after the lymphocyte transformation rate experiment is finished, 1mL of mixed solution of 1% chicken erythrocyte physiological saline is injected into the abdominal cavity, the abdomen of the mouse is lightly pressed, the mouse is killed by a cervical dislocation method after 30min, 2mL of PBS (phosphate buffer solution) is used for washing out abdominal macrophages, the washing-out solution is placed on a glass slide, washing and fixing are carried out, 100 macrophages are counted in each piece under a light microscope, and the number of cells phagocytosing the chicken erythrocytes is counted.

Percent (%) phagocytosis is the number of macrophages that phagocytose chicken red blood cells per 100 macrophages × 100%, and photographed.

The phagocytosis index a is the total number of chicken red blood cells phagocytosed from 100 macrophages/100 macrophages engulfing chicken red blood cells.

2.3 determination of mouse immune organ index

And (3) on the 7 th day, carrying out subcutaneous injection administration for about 6-8 h (namely in the process of a phagocytosis function experiment of abdominal macrophages), dislocating and killing the mice, washing out the abdominal macrophages, and weighing the thymus and the spleen.

Immune organ index (mg) organ mass/mouse mass (g)

Statistically processing the obtained experimental data to obtain the data

And (4) showing. The comparison between groups is performed by analysis of variance, and the comparison between two groups is performed by t test. P <0.05 is statistically significant.

2.4 results of the experiment

2.4.1 Effect of S-cyanomethyl-L-cysteine (SCC) on the immune organs of mice

TABLE 1

Note: denotes P <0.05, difference statistically significant compared to the placebo group.

As a result: as can be seen from the data in Table 1, the spleen index and the thymus index of the mice were significantly increased in the SCC medium dose group (10mg/kg) (P <0.05), the thymus index of the mice was significantly increased in the SCC low dose group (5mg/kg), and the effect of the high dose group (20mg/kg) on the spleen index and the thymus index of the mice was not statistically significant (P >0.05) as compared with the control group.

2.4.2 Effect of S-cyanomethyl-L-cysteine (SCC) on splenic lymphocyte proliferation in mice

TABLE 2

As a result: from the data in Table 2, it can be seen that the proliferation rate of lymphocytes in mice can be increased in each of the SCC dose groups compared with the blank control group, and the high dose group has the highest proliferation rate of lymphocytes (P <0.05) and has the dose effect.

2.4.3 Effect of SCC on mouse macrophage Capacity

TABLE 3

As a result: from the data in Table 3, it can be seen that the differences were statistically significant (P <0.05) in the SCC high dose group compared to the control group. SCC was shown to increase phagocytic capacity of mouse macrophages, and this effect was also confirmed from fig. 1.

Inhibition of HepG2 cells by S-cyanomethyl-L-cysteine (SCC)

3.1CCK-8 method for detecting the activity of lymphocytes in immune microenvironment after SCC treatment

HepG2 cells were plated at 2.0X 10⁴one/mL of the density was inoculated in a 96-well plate. After the cells are attached, the lymphocytes are added in the ratio of effective target ratio (lymphocytes: HepG2 ═ 100: 1). The cells were divided into a blank control group and each concentration group of SCC (10. mu.M, 30. mu.M, 100. mu.M), and each group was plated in 5 duplicate wells. SCC was added to each concentration group of SCC, and the same volume of PBS was added to the blank control group, which was then incubated at 37 ℃ with 5% CO₂And after 24 hours in a 95% humidity incubator, measuring the growth inhibition rate of HepG2 cells by using a CCK-8 method, namely, indicating the activity condition of the lymphocytes under the condition.

Cell viability ═ 100% (OD value in experimental group/OD value in control group) ×

The cell growth inhibition rate (1-OD value of experiment group/OD value of control group) × 100%

As a result: referring to fig. 2, treatment with different doses of SCC resulted in lymphocyte binding to HepG2 cells, which resulted in decreased survival, increased inhibition, and dose-response of HepG2 cells, with highest inhibition of HepG2 cells by high doses of SCC. The experimental result shows that SCC can promote the apoptosis of HepG2 cells by promoting the activation of lymphocytes and inhibiting the proliferation of the HepG2 cells.

3.2 Rui's-Giemsa staining method for detecting lymphocyte activation rate after SCC treatment

HepG2 cells at 4X 10⁷The cells are inoculated into a six-well plate, and after the cells adhere to the wall, splenic lymphocytes are taken according to the proportion of 5 multiplied by 10⁹adding/L of the suspension into a six-hole plate, adding SCC (with final concentration of 1 mu M, 10 mu M and 30 mu M respectively) with different doses into an experimental group, culturing for 48h, taking out 100 mu L of a culture solution smear containing suspension cells, naturally drying, dripping Wright-Giemsa working solution, standing for 8min at room temperature, washing and drying with distilled water, observing and calculating the cell conversion rate under a light microscope, namely counting 100 lymphocytes per slice under the microscope, calculating the number of cells with medium conversion and above (larger than small lymphocytes, about 10-30 mu M in diameter, compact nuclear staining and appearance of nucleolus or 1-2 nucleolus), and countingCalculating the lymphocyte transformation rate.

As a result: referring to FIGS. 3 and 4, each of the SCC concentration groups activated splenic lymphocytes of mice as compared with the blank control group. The swiss-giemsa staining method is used for staining and observing the lymphocytes, the activation rate of the lymphocytes is detected, the number of the lymphocytes after the SCC treatment and the activation rate are obviously increased, and 1 mu M is the optimal concentration (P < 0.01). This experiment demonstrates that SCC induces apoptosis by promoting lymphocyte activation, inhibiting HepG2 cell proliferation, and has a dose effect.

3.3 detection of expression of PD-1 in lymphocytes by Western blot method

HepG2 cells at 4X 10⁷The cells are inoculated into a six-well plate, and after the cells adhere to the wall, splenic lymphocytes are taken according to the proportion of 5 multiplied by 10⁹adding/L into a six-well plate, adding different doses of SCC (to make the final concentration be 1 mu M, 10 mu M and 30 mu M respectively) into an experimental group, culturing for 48h, sucking the culture solution containing the suspended lymphocytes into a 1.5mL centrifuge tube, centrifuging for 10min at 1500 r.min < -1 > to obtain lymphocyte precipitates, adding 80 mu L of lysis solution (lysis buffer) into each centrifuge tube to lyse the cells, and lysing the cells in the centrifuge tube on a shaker for at least 30 min. Then, the mixture was centrifuged at 13190rpm at 4 ℃ for 10min, and the supernatant was collected. And (3) determining the protein concentration by using a BCA kit, determining the absorbance at the wavelength of 562nm by using a microplate reader, drawing a protein standard curve, respectively calculating the concentration of each group of proteins, and calculating the volume of the protein to be added according to the protein concentration and the amount of the protein to be added.

The determined proteins were loaded in equal amounts and subjected to polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred to nitrocellulose membranes (PVDF). Sealing with 5% skimmed milk powder on shaking table for 1 hr. The PD-1 primary antibody and the beta-actin are placed on ice to be incubated for 1h by a shaking table and placed in a refrigerator at 4 ℃ overnight. Washing the membrane with TBST × 1 for 5min 3 times, slowly incubating with a goat anti-rabbit (1:1000) secondary antibody at room temperature in a shaking table for 1h, washing the membrane with TBST × 1 for 5min 3 times, detecting with ECL, developing, fixing, and scanning for observation.

As a result: referring to FIG. 5, the expression level of PD-1 protein on the surface of lymphocytes was lowest (P <0.01) after treatment with 1. mu.M SCC, whereas as the concentration of SCC increased, PD-1 expression increased and lymphocyte activation decreased. The SCC can block an immune escape mechanism of tumor cells by down-regulating the expression of PD-1 protein, so that the secreted PD-1 ligand (PD-L1) is reduced in binding with the PD-1 on the surface of the lymphocyte, and the lymphocyte is activated to generate an immune response reaction.

3.4 RT-qPCR detection of mRNA expression of PD-L1 in PD-1 and HepG2 cells in lymphocytes

Taking HepG2 according to 4X 10⁴The cells are inoculated into a 6-well plate in a concentration mode, lymphocytes are added according to the step 3.2 after the cells are attached to the wall and are arranged into groups, the lymphocytes and HepG2 cells are separated after being cultured for 48 hours, total RNA is extracted by respectively using an RNAiso Plus kit (TaKaRa company, batch number: 9108), the concentration and the quality of the RNA are measured by a micro ultraviolet spectrophotometer, and Prime Script is used after purity determination and quantification^TMRT reagent kit Kitwithg DNA Eraser kit (TaKaRa company, batch number: RR047A) to obtain cDNA, using the cDNA as a template to perform RT-qPCR amplification, and using GADPH as an internal reference gene. The procedure for amplifying PD-1, PD-L1 was as follows: 30s at 94 ℃; 94 ℃ for 5 s; 30s at 60 ℃; 39 Cycles. The primer sequences are shown in Table 4 (the sequences are shown in SEQ NO: 1-6).

TABLE 4 primer sequences

As a result: referring to FIG. 6, the expression level of PD-1mRNA on lymphocyte surface was lowest (P <0.01) after treatment with 1 μ M SCC, while the expression of PD-1 was increased and the activation of lymphocytes was decreased with the increase of SCC concentration, consistent with the Western blot results.

Referring to fig. 7, expression of PD-L1 in HepG2 cells significantly decreased the expression level of PD-L1 mRNA in HepG2 cells after treatment with 1 μ M SCC compared to the blank control group (P < 0.01).

The above results show that SCC can reduce the expression of PD-1, PD-L1 mRNA, reduce the expression of PD-1, PD-L1 protein, lead to the reduction of the binding of PD-1 ligand (PD-L1) secreted by HepG2 and the PD-1 on the surface of the lymphocyte, thus activating the lymphocyte and generating immune response reaction.

Application of S-cyanomethyl-L-cysteine (SCC) and quinoline derivative of N-isosterin (New Tecotorigenin) in combination for detecting inhibition rate of lymphocytes and HepG2 cells

HepG2 cells at 4X 10⁷The cells are inoculated into a six-well plate, and after the cells adhere to the wall, splenic lymphocytes are taken according to the proportion of 5 multiplied by 10⁹the/L was added to six well plates and the milder drug concentration (SCC: 10. mu.M, New Tecotorigenin: 2. mu.g/mL) was selected, different ratios of SCC: new Tecotorigenin (0, 10: 0, 0: 2, 10: 2), after co-culturing for 48h under the same conditions, was observed under an inverted microscope and photographed for recording, respectively.

The results show that: referring to fig. 8, the inhibition rate of HepG2 was 17% when treated with 10 μ M SCC alone. The inhibition of HepG2 was 31% when treated with New Tectorigenin alone. When SCC and New Tectrorigenin were combined, the inhibition rate of HepG2 cells was 66%. The results show that when SCC and New Tecotorigenin are combined to treat lymphocytes and HepG2 cells, the number of HepG2 cells is obviously reduced, and the inhibition rate of HepG2 cells is increased (P <0.01), which indicates that when SCC and New Tecotorigenin are combined, SCC can enhance the cancer inhibition effect of New Tecotorigenin by activating lymphocytes.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

SEQUENCE LISTING

<110> Guangzhou university

<120> PD-1/PD-L1 inhibitor and application thereof

<130> 2020.10.14

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213> GAPDH upstream primer

<400> 1

agtgccagcc tcgtctcata 20

<210> 2

<211> 20

<212> DNA

<213> GAPDH downstream primer

<400> 2

gatggtgatg ggtttcccgt 20

<210> 3

<211> 21

<212> DNA

<213> PD-1 upstream primer

<400> 3

ccccaaggaa aaatcgagga g 21

<210> 4

<211> 21

<212> DNA

<213> PD-1 downstream primer

<400> 4

gggcgagggg ctgggatatc t 21

<210> 5

<211> 20

<212> DNA

<213> PD-L1 upstream primer

<400> 5

ggtgccgact acaagcgaat 20

<210> 6

<211> 20

<212> DNA

<213> PD-L1 downstream primer

<400> 6

agccctcagc ctgacatgtc 20

Claims

1. A PD-1/PD-L1 inhibitor, comprising S-cyanomethyl-L-cysteine.

2. A PD-1/PD-L1 inhibitor comprising a quinoline derivative of S-cyanomethyl-L-cysteine and N isostere tectoridin.

3. The inhibitor according to claim 1 or 2, wherein the concentration of S-cyanomethyl-L-cysteine is 1 to 100 μ M.

4. The inhibitor according to claim 1 or 2, wherein the S-cyanomethyl-L-cysteine is prepared by a process comprising the steps of:

5. The inhibitor according to claim 4, wherein in the step S1, the stirring temperature is-10 to 10 ℃.

6. The inhibitor as in claim 4, wherein in step S2, the filtrate is recrystallized from a water-ethanol solution with a volume ratio of (1-3) to (2-4).

7. Use of the inhibitor of claim 1 or 2 in the preparation of a medicament or formulation against liver cancer.

8. The use according to claim 7, wherein the S-cyanomethyl-L-cysteine is released by liberating H₂S promotes the activation of lymphocytes, down-regulates the expression of PD-1 gene, down-regulates the expression of PD-L1 gene in liver cancer cells, and inhibits the PD-1/PD-L1 pathway, thereby promoting the apoptosis of the liver cancer cells.

9. The use of claim 7, wherein S-cyanomethyl-L-cysteine promotes apoptosis of N-isosteric tectoridin quinoline on hepatoma cells.

10. The use of claim 7, wherein the liver cancer cells comprise HepG2 cells.