A compound BP220805 with anti-tumor effect and its preparation method and application

Abstract

The present invention discloses a compound BP220805 with anti-tumor effect and its preparation method and application, and relates to the field of biomedical technology. The chemical structural formula of the compound BP220805 is: The present invention also provides the application of the compound BP220805 in the preparation of PD-1 inhibitors or anti-tumor drugs. The compound BP220805 can effectively block the PD-1 / PD-L1 interaction, not only can it increase the T cell infiltration activity, but also can change the tumor immune microenvironment, reactivate the immune response that is beneficial to anti-cancer, thereby effectively inhibiting the growth of tumors.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a compound BP220805 having an anti-tumor effect, and a preparation method and application thereof. Background Art

[0002] At present, the treatment methods for cancer usually include surgery, radiotherapy and systemic therapy (chemotherapy, hormone therapy, targeted biological therapy). Several treatment methods have their advantages and disadvantages. Surgery is not suitable for malignant lymphoma, non-solid tumors that spread throughout the body, and patients who cannot undergo surgical treatment; radiotherapy is not suitable for tumors that are not sensitive to radiation, and the various complications and high costs it brings are also difficult for patients to accept; chemotherapy is the patient's tolerance to drugs and its high side effects cause irreversible damage to the patient's body. Cancer immunotherapy has brought new hope to cancer patients. Cancer immunotherapy achieves the purpose of treatment by mobilizing the body's own immune system to resist or even kill tumors. In view of the fact that cancer immunotherapy has achieved long-term and lasting therapeutic effects in many advanced and metastatic solid cancers, it has triggered in-depth basic and translational research on the mechanism of cancer-immune cell interaction.

[0003] PD-1 / PD-L1 immune checkpoint inhibitory antibodies are already in clinical use. PD-1 / PD-L1 antibodies have poor tumor penetration, are extremely unevenly distributed in tumor tissues, have low patient immune response rates, and can only be administered intravenously. Compared with monoclonal antibody drugs, small molecule compounds (including small molecule peptide compounds) are easily absorbed, have high oral bioavailability, and have relatively low immune-related adverse reactions (irAEs). Therefore, it is urgent to develop small molecule inhibitors targeting the PD-1 / PD-L1 immune checkpoint. Summary of the invention

[0004] The purpose of the present invention is to provide a compound BP220805 with anti-tumor effect and its preparation method and application, so as to solve the problems existing in the above-mentioned prior art. The compound BP220805 can effectively block the PD-1 / PD-L1 interaction, not only can it increase the T cell infiltration activity, but also can change the tumor immune microenvironment (TIME), reactivate the immune response that is beneficial to anti-cancer, and thus effectively inhibit the growth of tumors.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a compound BP220805 having an anti-tumor effect, and the chemical structural formula of the compound BP220805 is as follows:

[0007]

[0008] The present invention also provides a method for preparing the compound BP220805, comprising the step of mixing sophoroside, bromopentane, potassium carbonate and N,N-dimethylformamide to obtain the compound BP220805.

[0009] Furthermore, the temperature of the mixed reaction is 80°C.

[0010] Furthermore, the mixing reaction time is 4 hours.

[0011] Furthermore, the reaction ratio of the sophoroside, the bromopentane, the potassium carbonate and the N,N-dimethylformamide is 5mmol:7.5mmol:10mmol:10mL.

[0012] The present invention also provides the use of the above-mentioned compound BP220805 in the preparation of PD-1 inhibitors.

[0013] The present invention also provides a PD-1 inhibitor, the active ingredient of which includes the above-mentioned compound BP220805.

[0014] The present invention also provides the use of the compound BP220805 in the preparation of anti-tumor drugs.

[0015] Furthermore, the tumor is colorectal cancer or melanoma.

[0016] The present invention also provides an anti-tumor drug, the active ingredient of which includes the above-mentioned compound BP220805.

[0017] The present invention discloses the following technical effects:

[0018] The present invention has developed a new, low-toxic isoflavone derivative, named BP220805, which can effectively block the PD-1 / PD-L1 interaction, not only improve the T cell infiltration activity, but also change TIME, reactivate the immune response that is beneficial to anti-cancer, and thus effectively inhibit the growth of tumors. In in vitro experiments, the present invention confirms that the compound BP220805 has a blocking effect on the PD-1 / PD-L1 protein interaction and can reduce the expression of PD-L1 on the surface of tumor cells; the compound BP220805 can directly inhibit the migration ability and clone formation ability of B16-F10 cells; in the co-culture system, BP220805 enhances the killing ability of in vitro T cells against tumor cells by upregulating the transcription levels of cytokines such as IL-2, IFN-γ, TNF-α and the cellular immune killer factor GZMB; in in vivo experiments, the present invention found that the compound BP220805 can significantly inhibit the growth of colorectal cancer and pigment tumor tissues, and significantly increase the proportion of CD4+T and CD8+T cells in tumor tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 is the chemical structure diagram of compound BP220805;

[0021] Figure 2 is the NMR carbon spectrum of compound BP220805;

[0022] Figure 3 is the H NMR spectrum of compound BP220805;

[0023] Figure 4 The figure is the result of molecular dynamics analysis of the compound that binds most stably to PD-1; wherein A is a schematic diagram of the binding of compound BP220805 to PD-1, in which the blue end of the protein strip model in the figure is the N-terminus, the red end is the C-terminus, and the sticks represent the Poisson-Boltzmann distribution of the surface charge of the BP220805 molecule, in which red represents negative charge and blue represents positive charge; B is the Poisson-Boltzmann distribution of the surface charge of the complex formed by the binding of PD-1 and SC220805, in which red represents negative charge and blue represents positive charge; C is the root mean square deviation of the heavy atom position change of the ligand bound to PD-1; D is the interaction energy between the ligand bound to PD-1 and PD-1, which is the sum of the Lanner-Jones potential energy and the Coulomb potential energy;

[0024] Figure 5 This is the result of HTRF detection of the blocking effect of compound BP220805 on PD-1 / PD-L1;

[0025] Figure 6 The figure is the result of in vitro toxicity analysis of compound BP220805 on Jurkat cells, melanoma cells B16-F10 and colon cancer cells CT26;

[0026] Figure 7 Figure 1 shows the colony formation of B16-F10 cells at different BP220805 concentrations (A) and the statistical graph of the number of clones (B);

[0027] Figure 8 The results of the cell scratch test (A) and the statistical diagram of the scratch healing degree (B); wherein S5 represents compound BP220805;

[0028] Fig. 9 Western Blot detection results (A) and expression statistics (B) of PD-L1 protein in B16-F10 cells at different BP220805 concentrations; S5 represents compound BP220805;

[0029] Fig.10 It is a statistical graph of tumor killing rate at different BP220805 concentrations;

[0030] Fig.11 The expression statistics of GZMB (A), IL-2 (B), TNF-α (C) and IFN-γ (D) in the co-culture system of compound BP220805 and T cells; wherein S5 represents compound BP220805; Coculture represents the co-culture of tumor cells and T cells;

[0031] Fig.12 It is a statistical chart of IFN-γ expression in Jurkat T cells; S5 represents compound BP220805; Co-culture represents co-culture of tumor cells and T cells;

[0032] Fig.13 The statistical graphs of tumor volume changes after 14 days of intraperitoneal administration in mice (A) and weight changes in mice (B);

[0033] Fig.14 The results of the BP220805 inhibition experiment on melanoma are shown in Figure 1. A is a picture of mouse melanoma at different time points after drug administration; B is the change in mouse tumor volume after drug administration; C is the survival curve of melanoma mice 10 days after drug administration; D is the change in mouse weight after drug administration;

[0034] Fig.15 The results of the BP220805 inhibition experiment on colorectal cancer are shown in Figure 1. A is a picture of mouse colorectal cancer tumors at different time points after drug administration; B is an anatomical picture of mouse colorectal cancer tumors; C is the change in mouse tumor volume after drug administration; D is the change in mouse weight after drug administration;

[0035] Fig.16 CD4 + T cells and CD8 + T cell detection results; A and B are CD4 + T cells and CD8 + T cell flow cytometry; C and D are CD4 + T cells and CD8 + The percentage of T cells in tumor tissue. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] The sophora japonica glycoside used in the following examples has the molecular formula C 21 H 20 O 10 , CAS No. 152-95-4, structural formula is as follows:

[0042]

[0043] Example 1

[0044] Preparation of small molecule compounds: Sophora japonica glycoside (2.21 g, 5.0 mmol), bromopentane (1.15 g, 7.5 mmol), potassium carbonate (1.40 g, 10.0 mmol) and N, N-dimethylformamide (10 mL) were added to a 25 mL single-necked flask equipped with a magnetic stirrer, and the mixture was reacted at 80°C for 4 hours. After the reaction, the reaction solution was poured into 50 mL of water, the pH was adjusted to neutral with acetic acid, and the mixture was filtered. The filter cake was washed with water and n-hexane, and recrystallized from ethanol to obtain a light brown solid pentyl-sophora japonica glycoside (1.15 g, 46%), referred to as compound BP220805, with the chemical structure as shown below: Figure 1 As shown, the NMR carbon spectrum is Figure 2 As shown, the hydrogen spectrum is Figure 3 shown.

[0045] Example 2

[0046] 1. Experimental Materials

[0047] Melanoma cell line B16-F10 and colon cancer cell line CT26 were purchased from ATCC; Jurkat cell line was purchased from Wuhan University Cell Bank.

[0048] 2. Experimental Methods

[0049] 2.1 Molecular docking

[0050] The present invention uses the ZINC compound structure mirror database, the small molecule peptide compound (di- to pentapeptide) structure database constructed using the ChemScript script, and the PD1 or PDL1 protein three-dimensional structure data downloaded from the Protein Data Bank (PDB), and uses the Molecular Operating Environment software for flexible molecular docking, and uses the London free energy algorithm for preliminary screening, and then uses the GBVI / WSA free energy algorithm to optimize the compound structure from the preliminary screening results and sort it. GBVI / WSAΔG calculates the binding free energy of the interaction between the ligand and the receptor by formula (1), where ΔE Coul , ΔE sol , ΔE vdW They correspond to electrostatic, solvent and van der Waals interactions, ΔSA is the solvent exposure area, c is the change in entropy, and α and β are AMBER force field training parameters. The docking results are saved as PDB format structure files for molecular dynamics analysis.

[0051] ΔG bind ≈c+α[2(ΔE Coul +ΔE sol ) / 3+ΔE vdW +βΔSA weighted ](1)

[0052] The more negative the score of the GBVI / WSAΔG function is, the stronger the interaction between the ligand and the receptor is. This function takes into account factors such as translational / rotational entropy, electrostatic effect, solvent effect, van der Waals effect, solvent exposure area, etc. The calculation results are relatively accurate and are often used for virtual drug screening and structure-activity relationship exploration.

[0053] 2.2 Cell culture

[0054] (1) Cell recovery: Take the cells out of the liquid nitrogen tank and immediately transfer them to a 37°C water bath to dissolve them. Place the dissolved cell suspension into complete culture medium preheated at 37°C (see Table 1 and Table 2 for the formula), centrifuge at 1000 rpm for 3 min, remove the supernatant, gently resuspend the cell pellet in complete culture medium, mix well, and place in a cell culture dish for culture.

[0055] (2) Cell passaging: When the cells have grown all over the culture flask, the original cell culture medium is removed, the cells are washed three times with DPBS (see Table 3 for the formula), 0.25% trypsin is added and the cells are digested in an incubator (37°C, 5% CO2) for 2 min, and the cells are allowed to fall off from the bottom of the culture dish. Complete culture medium is added to stop digestion, and the cells are gently blown to fall off. The cells are transferred to a centrifuge tube and centrifuged at 1000 rpm for 3 min (for suspended cells, the cells in the culture flask are directly transferred to a centrifuge tube for centrifugation, and the centrifugation parameters are: 1000 rpm, 10 min), the supernatant is removed, the cells are resuspended with complete culture medium, inoculated into a new culture dish at an appropriate ratio, and placed in a 37°C incubator with 5% CO2 for continued culture.

[0056] (3) Cell cryopreservation: First, prepare the cryopreservation solution. The volume ratio of DMEM, FBS and DMSO is 4:4:2. Do not mix FBS and DMSO first. The cryopreservation solution should be prepared and used immediately. Select cells in very good condition for cryopreservation. Wash and digest them according to the cell passaging method. Collect the cells and resuspend the cell pellet in complete culture medium (suspended cells need to be counted and resuspended to make the cryopreserved cell concentration 6×10 6 / mL), add the cell suspension and freezing solution into the cryotube at a volume ratio of 1:1, put the cryotube into a cryobox precooled at 4℃, place it at 4℃ for half an hour, transfer it to -20℃ for 4 hours, and transfer it to a liquid nitrogen tank for long-term storage after being stored at -80℃ overnight.

[0057] Table 1 DMEM cell culture medium formula (pH 7.0)

[0058]

[0059] Table 2 RPMI 1640 cell culture medium formula (pH 7.2)

[0060]

[0061] Table 3 DPBS formula (pH 7.0)

[0062]

[0063] 2.3 Western Blot Experiment

[0064] Obtaining protein samples: After the cells reach the treatment time, take out the cells and place them on ice, discard the culture medium, and wash them three times with pre-cooled PBS; add cell lysis solution (formula see Table 4), gently scrape the cells with a cell scraper, transfer to a 1.5mL EP tube, lyse on ice for 15min, ultrasonically disrupt, 30% power, ultrasonic for 2s, stop for 5s, and ultrasonic for a total of 30s. Put the broken cell suspension into a pre-cooled 4℃ centrifuge, 12000rpm, 15min, and carefully transfer the supernatant to a new centrifuge tube after centrifugation to obtain a protein sample. After determining the protein concentration, add protein loading buffer, and denature the protein in a metal water bath at 98℃ for 5min for SDS-PAGE electrophoresis.

[0065] Table 4 Cell lysis solution formula (pH 7.0)

[0066]

[0067] (2) Determination of protein concentration: Dilute 2 mg / mL BCA protein standard solution to 0.2 mg / mL with deionized water and place on ice for later use. Prepare standard protein solutions of different concentrations by diluting according to the table below (Table 5). At the same time, dilute the protein sample 25 times, add 100 μL of each protein standard solution or protein sample dilution to a 96-well plate, and add two replicate wells for each concentration. Prepare the working solution at a ratio of BCAReagent A: BCAReagent B = 100:1, add 100 μL to each well, and mix immediately. Incubate at 37°C for 1 hour, cool to room temperature, and measure the absorbance of each well at 562 nm using an enzyme reader. Draw a standard curve and calculate the concentration of each protein sample according to the regression equation.

[0068] Table 5 Dilution of BCA standard solution

[0069]

[0070] (3) SDS-PAGE gel electrophoresis: Prepare 10% separation gel and concentration gel according to the following table (see Table 6). After the gel is completely polymerized, protein is loaded. The amount of protein per well is 50 μg. The same volume and mass are loaded per well of the same gel. After loading, electrophoresis is performed at a constant voltage of 80 V. When the indicator front reaches the bottom of the gel, the electrophoresis is stopped.

[0071] Table 6 SDS-PAGE gel formula

[0072]

[0073] (4) Transfer: After the electrophoresis is completed, pry open the glass plate, carefully remove the gel, measure the size and transfer the gel to the transfer solution. Cut the 0.45 μm nitrocellulose (NC) membrane and filter paper of appropriate size according to the measurement data, and soak them in the transfer solution. Perform semi-dry transfer in the order of filter paper, NC membrane, gel, and filter paper from bottom to top, and transfer at a constant voltage of 20 V for 1 hour.

[0074] (5) Blocking: Place the transferred membrane in TBST and wash three times, 10 min each time. Then place it in the previously prepared blocking solution and block it at room temperature on a shaker at 75 rpm for 2 h.

[0075] (6) Antibody incubation: After blocking, dilute the primary antibody in proportion with blocking solution, place the NC membrane in the primary antibody, incubate at 4°C overnight, and wash the membrane three times with TBST on the next day, 10 min each time; similarly dilute the secondary antibody with blocking solution, place the NC membrane in the secondary antibody, incubate at 37°C for 2 h, and wash the membrane three times with TBST, 10 min each time.

[0076] (7) HRP color development and detection: Prepare ECL luminescent solution in a dark place. Prepare solution A and solution B in a 1:1 ratio and drop them onto the NC membrane. Use Amersham Imager 800 ultra-sensitive multifunctional imager for development and storage. Use Image J to analyze the grayscale value of the bands.

[0077] (8) Preparation of relevant reagents:

[0078] TBST: 8 g / L NaCl, 2.42 g / L Tris, adjust the pH to 7.4 with concentrated hydrochloric acid, and add 1 mL / L Tween 20.

[0079] PBS: 8g / LNaCl, 0.2g / LKCl, 1.44g / LNaHPO4, 0.24g / LKH2PO4, hydrochloric acid adjusted the pH to 7.4.

[0080] Blocking solution: Add 2 g skim milk powder to 40 mL TBST and mix well.

[0081] 6× protein loading buffer: 4× Tris·Cl / SDS 70%(v / v)pH 6.8, glycerol 30%(v / v), SDS10%(v / v), 0.6M DTT, bromphenol blue 0.012%(w / v)

[0082] 5×SDS-PAGE gel running buffer: 1g / L SDS, 3g / L Tris, 144g / L glycine

[0083] Transfer solution: 5.82 g / L Tris, 2.93 g / L glycine, 0.376 g / L SDS, 200 mL / L methanol.

[0084] 10% ammonium persulfate: weigh 0.1 g ammonium persulfate powder and dispense it into 1.5 mL EP. Add 1 mL ddH2O when using.

[0085] 2.4CCK-8 cytotoxicity assay

[0086] Collect logarithmic phase cells, count and adjust the cell suspension concentration to 5×10 3 cells / well, plated in 96-well plates. After the cells adhered to the wall, they were stimulated with 7 different concentrations of compounds, and zero wells and control wells were set at the same time. Six replicate wells were treated for each treatment and cultured in an incubator at 37°C and 5% CO2. After 24h, 48h and 72h, 10μL CCK-8 solution was added to each well of the 96-well plate, incubated in an incubator at 37°C and 5% CO2 for 2h, and the optical density was measured at 450nm using an enzyme reader.

[0087] Cell survival rate = (As-Ab) / (Ac-Ab)×100%, where As is the optical density of the experimental well; Ac is the optical density of the control well; and Ab is the optical density of the blank well.

[0088] According to the cell survival rate at different drug concentrations, the half inhibitory concentration (IC) of the compound was calculated using GraphPad Prism 8 software. 50 value.

[0089] 2.5 Cell scratch assay

[0090] Digestion and centrifugation were performed to collect target cells, and then 5 × 10 5 =Add the target cells to the 6-well plate at a ratio of cells / well; (make sure that the 6-well plate is fully covered after the cells adhere to the wall, with no cell gaps). Use a marker pen to evenly draw horizontal lines on the back of the 6-well plate with a ruler, about every 0.5 to 1 cm, and draw a line across the hole. At least 5 lines are crossed in each hole of the 6-well plate. After the cells adhere to the wall, use a small white gun tip to measure the ruler and try to scratch it vertically to the horizontal line on the back. The gun tip should be vertical and evenly applied without tilting. Wash the cells 3 times with sterile PBS (add when attached to the wall, suck out when attached to the wall) to remove the scratched cells. Add serum-free medium for culture and place in a 37°C 5% CO2 incubator for culture. Take pictures at fixed positions at 0, 12 and 24 hours to record the cell healing status. Image J counts the cell migration distance.

[0091] 2.6 Cell clone formation experiment

[0092] Take cells in logarithmic growth phase, digest with 0.25% trypsin and gently blow to make them into single cells, count live cells, resuspend the cell pellet with DMEM culture medium containing 20% ​​fetal bovine serum, and dilute according to experimental requirements so that there are 500 cells in each well (6-well plate). Culture in a 5% CO2 incubator at 37°C for 1-2 weeks. Stop culturing when the cells grow into a single, clear clone visible to the naked eye. Remove the culture medium, wash twice with PBS, immerse in 4% paraformaldehyde for 15 minutes, and air dry appropriately. Stain with 0.1% crystal violet staining solution for 10 minutes, and wash off the crystal violet on the surface with double distilled water. Let dry, and photograph the entire well under a microscope to see the clone.

[0093] 2.7 T cell killing assay

[0094] Jurkat cells in the logarithmic growth phase were taken and stimulated with 2μg / mL phytohemagglutinin (PHA) for 48h to activate the cells. Cancer cells in the logarithmic growth phase were taken and plated in 96-well plates at 5000 cells / well. After they adhered to the wall, Jurkat cells were added to the 96-well plates at different effector-target ratios (5:1, 10:1, 20:1). Compounds of different concentrations were added and co-cultured for 48h. The suspended Jurkat cells were washed with PBS, replaced with new culture medium, and 10μL CCK-8 solution was added. After incubation at 37℃ for 2h, the absorbance value was measured at 450nm by an enzyme reader. Experimental groups: the blank group was unstimulated Jurkat cells; the PHA pretreatment group was Jurkat cells stimulated with 2μg / mL PHA for 48h; the co-culture group was stimulated Jurkat cells and cancer cells; the drug-treated group was a co-culture group treated with different drugs.

[0095] The relative apoptosis rate of cancer cells (%) = 1-[(As-Ab) / (Ac-Ab)]×100%, where As: absorbance of the experimental well; Ac: absorbance of the control well; Ab: absorbance of the blank well.

[0096] 2.8 ELISA method to detect the secretion level of related cytokines in the supernatant of the co-culture system

[0097] In the above-mentioned Jurkat effector T cell and tumor cell co-culture model established with an effector-target ratio of 10:1, BP220805 compound was added to a final concentration of 10 μM, αPD-1 (concentration of 1 μM) was used as a positive control, and an equal volume of DMSO was used as a negative control. The supernatant was collected after 48 hours of culture. The ELISA kit instructions were followed.

[0098] 2.9 Real-time PCR detection of cytokine transcription

[0099] (1) In the above-mentioned Jurkat effector T cell and tumor cell co-culture model established with an effector-target ratio of 10:1, BP220805 compound was added to a final concentration of 10 μM, αPD-1 (concentration of 1 μM) was used as a positive control, and an equal volume of DMSO was used as a negative control. The cells were cultured for 48 hours, and the Jurkat cells in the supernatant were collected. The samples were collected by Trizol, and the cells were lysed at room temperature for 10 minutes, and total RNA was extracted and reverse transcribed into cDNA using the SPARKscriptⅡRT Plus kit.

[0100] Real-time PCR was used to quantitatively detect the expression of cytokines. Sybr-green staining showed the fluorescence signal of the amplified product, β-actin was used as an internal reference, and the relative expression (RQ, relative quantity) was calculated according to the number of amplification cycles (CT value).

[0101] Real-time PCR primers are shown in Table 7.

[0102] Table 7 Real-time PCR primers

[0103]

[0104]

[0105] 2.10 Homogeneous Time-Resolved Fluorescence Technology

[0106] This experiment used PD-1 / PD-L1 Binding Kits to screen small molecule inhibitors that can block PD-1 / PD-L1 interaction. The reagents contained in PD-1 / PD-L1 Binding Kits are: Tag1-PD-L1, Tag2-PD-1, PD-1 / PD-L1Standard, Anti-Tag1 Eu Cryptate reagent, Anti-Tag2 XL665 antibody and PPIEuropium Detection Buffer.

[0107] HTRF detection principle: Anti-Tag1 Eu Cryptate reagent (donor) and Anti-Tag2XL665antibody (acceptor) are used to detect the interaction between Tag1-PD-L1 and Tag2-PD-1. When PD-L1 and PD-1 are tightly bound, the donor and the acceptor are brought close together. When the donor is excited by 320nm excitation light, the emitted fluorescence will be absorbed by the acceptor as its excitation light, and the acceptor will emit 665nm fluorescence. This phenomenon is called fluorescence resonance energy transfer. When the binding of PD-L1 and PD-1 is blocked by small molecule inhibitors, the distance between the donor and the acceptor becomes larger. When the donor is excited by 320nm excitation light, fluorescence resonance energy transfer will not occur at this time, and only the 620nm fluorescence emitted by the donor can be detected. According to the ratio of Signal 665 and Signal 620, small molecule inhibitors with better blocking effects are screened out.

[0108] Experimental procedure: Prepare a 96-well plate, add 4 μL of 1×Tag1-PD-L1 solution to each well, dilute the compound to 1 mM with PPI Europium Detection Buffer, and then dilute it step by step, add 2 μL to each well (2 μL buffer is added to the positive control and negative control groups), add 4 μL of 1×Tag2-PD-1 solution (4 μL buffer is added to the negative control), and incubate at room temperature for 15 min. Add 1:1 pre-mixed Anti-Tag1 Eu and Anti-Tag2 XL665 to the 96-well plate, add 10 μL to each well, and treat three replicates for each treatment. Seal the plate and incubate at room temperature for 2 h. With the excitation light at 320 nm, use a full-wavelength microplate reader to measure the signal values ​​at 665 nm and 620 nm.

[0109] 2.11 Mouse tumor formation experiment

[0110] (1) Preparation of reagents: Compound BP220805 was dissolved in DMSO, filtered through a 0.22 μm filter membrane, and then 40% PEG300, 5% Tween-80, and 45% saline were added in a volume ratio. The solution was ultrasonically shaken to make it clear and transparent, and then placed at room temperature for later use.

[0111] (2) Mice breeding: Four-week-old male Balb / c mice and male C57BL / 6 mice were purchased from the Guangzhou Experimental Animal Center. They were allowed to eat and drink freely, and the bedding was changed every two days. The average weight of the mice was about 20 g after 5-8 weeks of breeding. The animal experiments of the present invention were in accordance with the "Regulations on the Administration of Experimental Animals" issued by the State Science and Technology Commission, and the experimental methods were approved by the Animal Ethics Committee of Hainan University.

[0112] (3) Tumor: CT26 cells were cultured and collected when they were in the logarithmic phase. The medium was changed one day before the cells were collected. After digestion and collection, the cells were washed with PBS and the cells were counted. 1×10 6 The cells / 0.1 mL / mouse were inoculated into the right hind limb of the mouse, and the growth of the tumor was observed.

[0113] (4) Drug administration: When the tumor grows to 100 mm 3 Around 2:00 pm, intraperitoneal administration was performed at 0.1 mL / 10 g, and the control group was given the same volume of solvent (DMSO: PEG300: Tween-80: saline = 10%: 40%: 5%: 45%). Administration was performed twice a day for a total of 14 days, and the weight and tumor size of the mice were measured before each administration.

[0114] (5) Tumor measurement and dissection: After the administration, the mice were anesthetized and killed, and the tumor tissues, livers, and kidneys of each mouse were dissected and weighed separately. The tumor volume of the mouse was measured, and the calculation formula was V = tumor long diameter × short diameter × short diameter / 2. After the measurement, the tumor and mouse organs were stored in paraformaldehyde for future use.

[0115] 2.12 Detection of CD8 in tumor tissue by flow cytometry + cell

[0116] The tumor tissues of each group of mice were minced in HBSS buffer, and the cells were collected by centrifugation and transferred to 2 mL of digestion solution (1 mg / mL collagenase I, dissolved in high-glucose DMEM medium in advance), and digested at 37°C for 90 min, with continuous slow rotation and mixing. Add 5 mL of DMEM medium containing FBS to terminate the digestion. Centrifuge at 2000 rpm for 7 min to remove the supernatant, wash once with DMEM, pipette 10 times, pass through a 70 μm filter, collect the filtered cells by centrifugation, resuspend in PBS and count. Adjust the cell concentration to 100 μL containing 1×10 6 cells, added anti-mouse CD3, CD4 and CD8 antibodies, incubated on ice for 30 min, washed twice with PBS, resuspended in 500 μL PBS, and detected by flow cytometry.

[0117] 2.13 Statistical analysis

[0118] All statistical analyses were performed using GraphPad Prism 8, and the results are presented as mean ± SEM. When comparing two groups, data were analyzed by student t test. Between multiple groups, one-way ANOVA analysis was performed using Tukey's test. Post hoc tests were performed only when F reached P < 0.05 and there was no significant heterogeneity of variance. P values ​​< 0.05 were considered statistically significant.

[0119] 3. Results and Analysis

[0120] 3.1 Molecular docking results

[0121] Molecular docking is a process of predicting the binding mode of protein-ligand through geometric matching and energy matching between two or more molecules, and identifying each other to find the best matching mode. It is of great significance in auxiliary drug screening and drug design. In 1894, Emil Fischer proposed the concept of molecular docking based on the "key theory", comparing the binding relationship between receptor and ligand to the relationship between key and lock, and inferred that there is a specific binding region between receptor and ligand, and this region has structural specificity, satisfying the shape complementarity of receptor-ligand in space. In the "key theory", the shape of the lock and key is fixed, but in the actual recognition process, the binding of ligand and receptor is constantly changing to meet the shape matching and energy matching. Koshland proposed the "induction theory" in 1958, believing that the change of receptor conformation in the binding pocket is forced by the induction of the ligand, so that a reversible complementary fit is formed between the two. In the past few years, several docking tools have been developed for molecular docking, such as LeDock, rDock, AutoDockVina, AutoDock, UCSF DOCK, GOLD, Glide, Surflex-Dock, LigandFit and MOEDock. Computer-aided drug design (CADD) technology uses computers to simulate the interaction process and degree of fit between protein receptors and drug ligand small molecules, which can avoid the time-consuming, labor-intensive and costly shortcomings of traditional drug screening methods. Using CADD technology, one or more small molecule ligands that are most likely to interact with the target protein can be quickly and accurately screened.

[0122] Molecular Operating Environment (MOE) software was used for flexible molecular docking, and the London free energy algorithm was used for initial screening. The GBVI / WSA free energy algorithm was then used to optimize the compound structures from the initial screening results and rank them.

[0123] The present invention selects the top 32 protein-ligand complex spatial conformations ranked by binding free energy in the molecular docking results after refinement analysis by GBVI / WSA algorithm, and uses molecular dynamics method to dynamically analyze their binding states in 0.1 M sodium chloride solution, thereby obtaining compounds that may stably bind to PD-1. Figure 4Figures C and D show some of the analysis results of the six compounds that bind most stably to PD-1. The root mean square deviation (RMSD) analysis of the position changes of the heavy atoms in the compound molecules showed that they bind relatively stably and do not detach from the PD-1 protein. Among them, compound SC220805 binds most stably to PD-1 ( Figure 4 C), the interaction energy is also the highest (negative energy represents mutual attraction) ( Figure 4 (D) The binding position of SC220805 to PD-1 is shown in Figure 4 As shown in A and B. It is reported that in the hydrophobic space, several hydrophilic residues of hPD-L1 interact with hPD-1 residues Asn66, Tyr68, Gln75, Thr76, Asp77, Lys78, Ala132 and Glu136 to form a large number of hydrogen bonds and salt bridges, among which Glu136 contributes the most. BP220805 of the present invention will compete with the ligand PD-L1 for binding to PD-1. When there is a sufficient amount of BP220805, the PD-L1 binding site on PD-1 will be blocked, and therefore, the PD-1 signaling pathway will not be activated.

[0124] 3.2 HRTF assay to detect the blocking effect of compounds on PD-1 / PD-L1

[0125] In this experiment, the present invention uses HTRF experiment to evaluate the blocking effect of compound BP220805 on PD-1 / PD-L1 binding in vitro. The results of the measurement are respectively used as the compound concentration and HTRF Ratio (665nm / 620nm×10000) fluorescence ratio for curve fitting. The results are shown in Figure 5 As shown in the figure, as the concentration of the compound increases, the HTRF Ratio gradually decreases, indicating that the compound can block the binding of PD-1 / PD-L1 and has a concentration-dependent effect. Its half-maximal inhibition concentration is 59.84 μM, indicating that BP220805 can effectively block the in vitro binding of PD-1 / PD-L1.

[0126] 3.3 Cytotoxic effect of compound BP220805

[0127] In order to understand the cytotoxicity of compound BP220805, the present invention used CCK-8 to conduct a series of in vitro cytotoxicity experiments on cells, including human T lymphocytes Jurkat, mouse skin melanoma cells B16-F10, and mouse colon cancer cells CT26. Figure 6 As shown, the toxicity to these three cell lines was very low, and the half inhibitory concentration IC 50 The values ​​(Table 8) were all greater than 30 μM, indicating that compound BP220805 could not inhibit the proliferation and growth of cells at low concentrations.

[0128] Table 8 Analysis of cytotoxicity of compound BP220805 on several cell lines by CCK-8 assay

[0129]

[0130] 3.4 Ability of compound BP220805 to inhibit cancer cell colony formation

[0131] The cell clone formation experiment can directly show the ability of the compound to form cancer cell colonies and their proliferation ability. By comparing the number and size of colony formation before and after administration of cancer cells, the present invention can find that compound BP220805 significantly inhibits the formation of B16-F10 melanoma cell colonies. With the increase of the administration concentration, the number of colonies is significantly reduced, and the size of the colonies is significantly reduced. At a concentration of 10 μM or more, the inhibition of compound BP220805 on the formation of B16-F10 melanoma cell colonies is significantly enhanced, and the size of the colonies also changes significantly ( Figure 7 ).

[0132] 3.5 Compound BP220805 inhibits cancer cell migration

[0133] The cell scratch test can analyze the migration ability of cells by the speed of scratch healing. When cells grow to a single layer, a blank area is artificially created on the single layer of cells, called a "scratch". The cells at the edge of the scratch will slowly enter the blank area, allowing the artificial scratch to heal. The migration ability of cells can be judged based on the area of ​​the scratch. Figure 8 As shown, with DMSO as the control group, after culturing B16-F10 melanoma cells for 12 hours, there was no significant difference in the scratch area between the 10 μM concentration of compound BP220805 and the control group, but at 24 hours, the scratch area of ​​the compound BP220805 group was significantly higher than that of the control group, indicating that compound BP220805 can significantly inhibit the migration ability of melanoma cells.

[0134] 3.6 Compound BP220805 reduces the expression of PD-L1 on tumor cells

[0135] The present invention uses HTRF to determine the blocking effect of BP220805 on PD-1 / PD-L1 and explores the direct killing effect of BP220805 on cancer cells. In addition to directly blocking the PD-1 / PD-L1 interaction, many small molecule compounds can also inhibit the PD-1 / PD-L1 signaling pathway by regulating the expression of PD-L1 in tumor cells. The present invention also found that when the drug concentration reaches 20 μM, the expression of PD-L1 in tumor cells can be downregulated ( Fig. 9BP220805 compound may jointly inhibit the PD-1 / PD-L1 signaling pathway by blocking the interaction of PD-1 / PD-L1 and downregulating the expression of PD-L1.

[0136] 3.7 Compound BP220805 upregulates the ability of T cells to kill cancer cells

[0137] The present invention found that compound BP220805 can block the mutual binding of PD-1 / PD-L1, has low cytotoxicity to cancer cells, and can inhibit the colony formation and migration ability of cancer cells. In order to evaluate the ability of BP220805 to restore the immune ability suppressed by the activated pathway, the present invention co-cultured B16-F10 and CT26 cancer cells with Jurkat T cells, such as Fig.10 As shown, compound BP220805 can enhance the killing effect of T cells on cancer cells, and the killing rate increases with the increase of compound BP220805 concentration. At a concentration of 20μM, the killing rate is as high as more than 90%, indicating that compound BP220805 can enhance the killing rate of tumors by enhancing T cell activity. Compound BP220805 promotes the killing effect of T cells on B16-F10 cells more than CT26 cells.

[0138] 3.8 Compound BP220805 restores T cell effector function

[0139] Next, the present invention uses real time-PCR to determine the expression of GZMB, IL-2, IFN-γ and TNF-α in the co-culture system. Cytokines can stimulate the activation and proliferation of T cells in the tumor microenvironment to enhance the killing effect of T cells on tumor cells. The results are shown in Fig.11 It can be found that compared with Jurkat T cells cultured alone, the transcription level of cytokines in Jurkat T cells will decrease significantly when co-cultured with cancer cells, among which IFN-γ decreases most significantly. When 10μM BP220805 compound is added to the co-culture system, the downregulation of the expression of these cytokines can be significantly reversed, indicating that the compound BP220805 can restore or even improve the activity of T cells.

[0140] 3.9 Effect of compound BP220805 on IFN-γ expression in Jurkat cells in vitro

[0141] Cytokines play an important role in immune response. It has been previously reported that blocking PD-1 / PD-L1 can promote the activity of cytotoxic lymphocytes by increasing the secretion of cytokines such as interferon-γ (IFN-γ). The present invention studied the effect of BP220805 on the production of cytokine IFN-γ in Jurkat T cells in an in vitro co-culture system ( Fig.12 ). The results showed that when Jurkat T cells were co-cultured with cancer cells, the secretion of IFN-γ decreased, while the BP220805 treatment group was able to significantly increase the expression of IFN-γ, which was consistent with the qPCR results. The compound BP220805 blocked the PD-1 / PD-L1 signaling pathway, thereby alleviating the inhibition of IFN-γ production in the tumor microenvironment.

[0142] 3.10 Compound BP220805 inhibits the growth of colorectal cancer tumors

[0143] In the previous discussion, the present invention has explored that the compound BP220805 can inhibit the binding of PD-1 and PD-L1, can inhibit the migration and proliferation of tumor cells in vitro, and can improve the killing ability of T cells against tumor cells by restoring the expression level of cytokines. Next, the present invention evaluates the in vivo anti-tumor effect of BP220805. First, the present invention established a CT26 tumor-bearing mouse model, such as Fig.13 As shown in Figure A, when 30 mg / kg was intraperitoneally administered, compound BP220805 showed an inhibitory effect on tumor growth, and when 60 mg / kg was intraperitoneally administered, compound BP220805 showed a significant tumor inhibitory effect, and during the treatment, there was no significant change in the weight of the mice ( Fig.13 (middle B).

[0144] 3.11 Compound BP220805 can inhibit the growth of colorectal cancer and melanoma and has a certain safety

[0145] Next, the present invention evaluated the inhibitory effect of BP220805 on melanoma. Fig.14 As shown in Figure 2, at a dose of 60 mg / kg, the growth of melanoma was significantly inhibited, and there was no significant change in the body weight of mice ( Fig.15 D). Due to the rapid growth of melanoma, all mice in the control group died after the 10th day of treatment. However, only 6 mice in the drug-treated group survived. Compound BP220805 increased the survival rate of melanoma mice ( Fig.15 C). After that, the present invention performed biochemical tests on the mouse serum. As shown in Table 9, there was no significant difference in AST, ALT, ALP and BUN between the administration group and the control group. The compound BP220805 did not cause severe inflammatory reactions in the liver and kidneys, indicating that the compound BP220805 had good safety in treating mice.

[0146] Table 9 Serum biochemical test of B16-F10 tumor-bearing mice

[0147]

[0148]

[0149] Note: AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; BUN: serum urea nitrogen.

[0150] Similarly, 14 days of drug treatment of CT26 tumor-bearing mice confirmed that compound BP220805 significantly inhibited the tumor growth of mouse colorectal cancer ( Fig.15 ), and the weight of mice also did not change significantly before and after administration. The present invention took the mouse serum for biochemical detection after the administration (Table 10), and found that the AST and ALT levels of the control group exceeded the normal range. The present invention speculated that the growth of the tumor may cause an inflammatory response in the mouse body, but the AST and ALT levels of the administration group were within the normal range, indicating that the drug treatment of the present invention has a certain alleviating effect on the inflammatory response caused by the tumor. And there is no significant difference in ALP and BUN compared with the control group, indicating that the drug treatment does not cause obvious liver and kidney toxicity, and the drug treatment has a certain safety.

[0151] Table 10 Serum biochemical test of CT26 tumor-bearing mice

[0152]

[0153] Note: AST: aspartate aminotransferase; ALT: alanine aminotransferase; ALP: alkaline phosphatase; BUN: serum urea nitrogen.

[0154] 3.12CD4 + T cells and CD8 + T cell levels in tumor tissue

[0155] After evaluating the growth inhibition of colorectal cancer and melanoma, the present invention decided to further analyze the immunomodulatory effect of the compound on colorectal cancer. After the treatment, the mouse tumors were removed and the tumor infiltrating lymphocytes (TIL) in the colorectal cancer tumor tissue were analyzed by flow cytometry. Fig.16 As shown, compared with the control group, the CD4 + T cells and CD8 + The proportion of T cells increased significantly, indicating that compound BP220805 can increase the level of T cell infiltration in tumor tissues and further promote anti-tumor immune response.

[0156] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A compound BP220805 that blocks the interaction between PD-1 and PD-L1, characterized in that: The chemical structural formula of the compound BP220805 is as follows:

2. Use of the compound BP220805 as claimed in claim 1 in the preparation of PD-1 / PD-L1 inhibitors.

3. A PD-1 / PD-L1 inhibitor, characterized in that: The active ingredient comprises the compound BP220805 described in claim 1.

4. An anti-tumor drug, characterized in that: The active ingredient comprises the compound BP220805 described in claim 1.

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