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Application of small-molecule inhibitor ML385 in inhibition of tumor cells PD-L1

A small molecule inhibitor, PD-L1 technology, applied in the field of biomedicine, to achieve the effect of easy mass production, anti-tumor activity and low cost

Inactive Publication Date: 2022-06-07
NANTONG UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

suggest that activation of PD-L1L2-SE may be due to altered signaling pathways

Method used

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  • Application of small-molecule inhibitor ML385 in inhibition of tumor cells PD-L1
  • Application of small-molecule inhibitor ML385 in inhibition of tumor cells PD-L1
  • Application of small-molecule inhibitor ML385 in inhibition of tumor cells PD-L1

Examples

Experimental program
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Effect test

preparation example Construction

[0075] (1) Preparation of protein samples. Prepare lysis buffer (containing 10 μL PMSF and 990 μL Lysis buffer) and place in an ice bath for later use; cells are washed with pre-cooled PBS and then transferred to a sterilized centrifuge tube, and the supernatant is discarded after centrifugation; cells are resuspended in lysis buffer containing PMSF , lysed on ice bath for 30min (shaker); 4°C, high-speed centrifugation for 15min, and completely aspirated the supernatant (protein). After mixing the protein with 5×Loading buffer, pipette at a ratio of 4:1, boil in a metal bath at 95°C for 5 min, and store at -20°C.

[0076] (2) gel electrophoresis and transfer membrane for color development. Boil the protein sample again at 105°C for 5 min; prepare 10% separating gel according to the formula of protein gel, prepare 5% stacking gel for the upper layer, and add 10 μL protein sample to each well; adjust the voltage of the electrophoresis apparatus to 80V, when the bromophenol blue...

Embodiment 1

[0099] To verify the effect of ML385 inhibitor in tumor cells, the selected target cells were breast cancer SUM159 and MDA-MB-231, respectively. First, recover the cells. The specific example is as follows: take out the cryovial from -80°C, directly immerse it in warm water at 37°C, and shake it to thaw as soon as possible (slow freeze and quick-dissolve); take out the cryovial from the 37°C water bath, add 1 mL of Complete medium, mix well; centrifuge at 1000 rpm for 3 min, discard the supernatant; add DMEM medium containing 10% FBS to resuspend the cells, and evenly inoculate in a 60 mm petri dish, 5% CO 2 , 37°C incubator for static culture; replace the culture medium the next day and continue the culture; when the cell density reaches 90%, trypsinize the cells, and determine the number of wells (6-well plate) according to subsequent experiments, and continue to culture; NRF2 Inhibitor treatment: ML385, the concentration of selective treatment was 0, 20, 50, 100 μM, and the...

Embodiment 2

[0101] In order to fully understand the molecular mechanism of PD-L1L2-SE super-enhancer regulation of PD-L1 and PD-L2, the present invention carried out a fine analysis of PD-L1L2-SE, and found a core region (hg19: Chr9: 5,498, 950–5, 499, 663). In order to fully understand the function of this DNA sequence, the present invention designs all possible sgRNAs, 22 sgRNAs in total. By establishing stable sgRNA cell lines respectively, the present invention found that sgRNA-22 had the greatest effect on the expression of PD-L1 ( figure 1 shown in A-D).

[0102] In order to further verify the result, the present invention further tested with quantitative RT-PCR, and found that the sgRNA-22 stable cell line can significantly down-regulate the expressions of PD-L1 and PD-L2 (such as figure 2 shown). The above results indicate that the DNA where sgRNA-22 is located is extremely important for the high expression of PD-L1 and PD-L2.

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Abstract

The invention discloses an application of a small-molecule inhibitor ML385 in the aspect of inhibiting tumor cells PD-L1. Furthermore, the ML385 inhibitor inhibits the function of PD-L1 in tumor cells and activates the anti-tumor immune function of T cells. The embodiment of the invention also provides an application of the small-molecule inhibitor ML385 in the aspect of inhibiting tumor cells PD-L1 and PD-L2. Furthermore, the ML385 inhibitor destroys the core site of PD-L1L2-SE identified by the NRF2 by inhibiting the combination of the NRF2 and the MAF, so that the expression of PD-L1 and PD-L2 is inhibited. The embodiment of the invention further provides a medicine for killing tumor cells, and the medicine is characterized by comprising the small-molecule inhibitor ML385. Furthermore, the medicine further comprises an anti-PD-L1 antibody or an anti-PD-L2 antibody. According to the embodiment of the invention, experiments prove that the small molecule inhibitor ML385 obviously inhibits the expression of PDL1 in breast cancer cells SUM159 and MDA-MB-231.

Description

technical field [0001] The invention belongs to the field of biomedical technology, in particular to the application of a small molecule inhibitor ML385 in inhibiting tumor cell PD-L1, and the application of a small molecule inhibitor ML385 in inhibiting tumor cell PD-L1 and PD-L2. Background technique [0002] In recent years, the treatment of breast cancer has achieved remarkable clinical results. The main treatment methods are surgery, radiotherapy and chemotherapy. However, the 5-year survival rate is generally low, especially for patients with advanced disease. With the development of tumor biology and immunology, cancer immunotherapy has become a new research hotspot in the field of tumor therapy in recent years. Immune cells and enhance the body's anti-tumor immune response, specifically remove tumor minimal residual lesions, inhibit tumor growth, and break immune tolerance. Tumor immunotherapy is to overcome the mechanism of tumor immune escape, thereby reawakening ...

Claims

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Application Information

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IPC IPC(8): A61K31/427A61K39/395A61P35/00A61P15/14C12Q1/6886C12Q1/686
CPCA61K31/427A61K39/39558A61P35/00A61P15/14C12Q1/6886C12Q1/686C12Q2600/106C12Q2600/158A61K2300/00C12Q2545/114C12Q2521/107
Inventor 范义辉史淙霖郑雯毛仁芳
Owner NANTONG UNIVERSITY
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