Preparation method of inhibitor targeting MAX-PD-L1 promoter specific binding motif and double-target inhibitor
By developing an inhibitor preparation method that specifically binds to the MAX-PD-L1 promoter motif, the problem of drug resistance caused by existing anti-PD-L1 inhibitors failing to block PD-L1 expression in tumor cells has been solved. This method achieves highly specific and high-affinity inhibition of tumor immune escape, thereby improving the efficacy of immunotherapy for lung adenocarcinoma.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- GENERAL HOSPITAL OF SOUTHERN THEATRE COMMAND OF PLA
- Filing Date
- 2025-12-25
- Publication Date
- 2026-04-24
AI Technical Summary
Existing anti-PD-L1 inhibitors cannot effectively block tumor cells from reactivating PD-L1 expression through upstream regulatory pathways, leading to drug resistance. Furthermore, existing inhibitors do not target the PD-L1 transcriptional activation process, failing to address the issue of multi-mechanism synergistic escape.
The method for preparing inhibitors that specifically target the MAX-PD-L1 promoter binding motif includes enriching chromatin DNA fragments from target cancer cells, screening for specific binding motifs, designing high-affinity DNA aptamers and small molecule compounds, optimizing their affinity and cell permeability, screening for highly active inhibitors using EMSA and SPR experiments, and preparing dual-target inhibitors that simultaneously target the MAX and JAK1 kinase domains.
It significantly inhibits tumor immune escape, enhances the killing rate of T cells against tumor cells, improves the response rate of immunotherapy for lung adenocarcinoma, and has high specificity and high affinity, while reducing off-target effects.
Smart Images
Figure CN121915040A_ABST
Abstract
Description
Technical Field
[0001] This application relates to the field of biomedical technology, specifically to a method for preparing an inhibitor that specifically targets the MAX-PD-L1 promoter binding motif and a dual-target inhibitor. Background Technology
[0002] Lung cancer is one of the malignant tumors with the highest incidence and mortality rates worldwide. The lung cancer mortality rate has been increasing rapidly in recent years, with non-small cell lung cancer accounting for 80% of all lung cancers. Traditional surgery, radiotherapy, chemotherapy, and targeted therapy have made progress, but the 5-year survival rate for lung cancer patients remains low.
[0003] Immunotherapy targeting PD-1 / PD-L1 represents a major breakthrough in lung cancer treatment. PD-1 is programmed death receptor 1, and PD-L1 is programmed death ligand 1. PD-L1 is an immunosuppressive molecule on the surface of tumor cells, inhibiting T cell activity and mediating tumor immune escape by binding to PD-1 on the surface of T cells. Current anti-PD-L1 monotherapy is only effective in some patients, primarily because the regulatory mechanism of PD-L1 expression is not fully understood, especially regarding endogenous transcriptional regulatory pathways.
[0004] MAX transcription factors regulate downstream gene expression by forming dimers with MYC or MAD: MYC-MAX activates transcription, while MAD-MAX inhibits transcription. MAX exhibits heterogeneity in its overall role in tumors, for example, exhibiting both tumor-suppressing and tumor-promoting functions in small cell lung cancer. However, current research has not revealed the impact of MAX on the regulation of PD-L1 transcription and immune escape. MYC is a transcription factor encoded by a proto-oncogene that activates PD-L1 transcription by forming a dimer with MAX. MAD is a MAX dimerized protein that dimers with MAX to inhibit transcription. MAX, a transcription factor and the core target of this study, activates PD-L1 transcription by binding to the PD-L1 promoter, thus promoting immune escape.
[0005] This research project, through analysis of the TCGA database, discovered a high correlation between the transcriptional gene MAX and PD-L1 mRNA in lung adenocarcinoma. ChIP-seq (chromatin immunoprecipitation sequencing) showed that the MAX transcriptional gene specifically binds to the promoter region of the PD-L1 gene. Functional experiments verified that the MAX transcriptional gene promotes PD-L1 transcription and expression, and inhibits T-cell killing of tumor cells. Survival analysis showed that high MAX / PD-L1 expression predicts poor prognosis (when the expression levels of MAX transcription factor and PD-L1 protein in the tumor tissue of lung adenocarcinoma patients are significantly higher than those in normal or low-expression individuals, patients have shorter survival times or faster disease progression). Based on these findings, the project proposes that MAX activates PD-L1 transcription by binding to a specific motif of the PD-L1 promoter, mediating immune escape in lung adenocarcinoma. Inhibitors targeting this motif can block PD-L1 expression at its source, thereby improving immune response.
[0006] Therefore, existing technologies have shortcomings and need further improvement and refinement. Summary of the Invention
[0007] Based on this, in order to solve the problems mentioned in the prior art, this application provides a method for preparing an inhibitor that specifically targets the MAX-PD-L1 promoter binding motif. By precisely targeting the MAX-PD-L1 signaling axis, it significantly inhibits tumor immune escape and has high specificity, high affinity and good prospects for clinical translation.
[0008] The first approach provided in this application is: a method for preparing an inhibitor that specifically binds to the MAX-PD-L1 promoter motif, comprising the following steps: Enrich chromatin DNA fragments in target cancer cells that directly bind to the transcription factor MAX protein, and obtain the sequence information of these chromatin DNA fragments; MAX binding peaks located in the promoter region of the PD-L1 gene were screened from the sequence information of chromatin DNA fragments, and the conserved sequences of the binding peaks were analyzed to preliminarily determine candidate motifs. Candidate motifs were subjected to site-directed mutagenesis, and the effect of the mutated transcription factor MAX on the transcriptional activity of the PD-L1 promoter was examined to determine the specific binding motif of MAX to the PD-L1 gene promoter region. Based on the specific binding motif, DNA aptamers with high affinity for transcription factor MAX were screened, and small molecule compounds were virtually screened using a molecular docking model between the DNA binding domain of transcription factor MAX and the specific binding motif. The inhibitory effects of DNA aptamers or small molecule compounds on the binding of transcription factor MAX to the PD-L1 promoter, the inhibitory effect of transcription factor MAX on the activation of the PD-L1 promoter, and the inhibitory effect on immune escape of target cancer cells were verified sequentially. Cholesterol modification was performed on validated DNA aptamers to improve cell permeability, and hydrogen bonding and hydrophobic interactions were optimized on the binding bags of small molecule compounds to improve affinity. The dissociation constants of DNA aptamers or small molecule compounds with transcription factor MAX were determined by SPR assay. High-affinity molecules with dissociation constants less than the first threshold were screened out. Furthermore, the inhibition rate of high-affinity molecules on the binding of specific binding motifs was detected by EMSA assay, and highly active inhibitors with inhibition rates greater than the second threshold were screened out.
[0009] Furthermore, the step of enriching chromatin DNA fragments in target cancer cells that directly bind to the transcription factor MAX protein and obtaining the sequence information of these chromatin DNA fragments includes: Chromatin DNA fragments that directly bind to the transcription factor MAX protein in lung adenocarcinoma cells were enriched using chromatin immunoprecipitation technology, and the sequence information of the chromatin DNA fragments was obtained by high-throughput sequencing.
[0010] Furthermore, the lung adenocarcinoma cells include A549 and H1975.
[0011] Further, the steps of screening for MAX binding peaks located in the PD-L1 gene promoter region from the sequence information of the autochromatin DNA fragments, and analyzing the conserved sequences of the binding peaks to preliminarily determine candidate motifs include: The sequence information of the autochromatin DNA fragment was compared with that of the human genome hg38. Visual analysis was performed using IGV software to screen out the MAX binding peak located in the promoter region of the PD-L1 gene. The conserved sequence of the binding peak region was analyzed using MEME Suite to preliminarily determine candidate motifs.
[0012] Furthermore, the PD-L1 gene promoter region extends from 2kb upstream to 500bp downstream of the transcription start site.
[0013] Furthermore, the step of performing site-directed mutagenesis on the candidate motif includes: For candidate motifs in the PD-L1 promoter region, complementary primers covering the mutant region were designed, with the mutant site located in the middle of the primer and restriction sites introduced at both ends. Using a plasmid containing the PD-L1 promoter as a template, high-fidelity DNA polymerase was used for PCR amplification to obtain a plasmid containing the mutation site. Methylated template DNA was digested with Dpnl restriction endonuclease, while unmethylated mutant plasmids were retained; The digestion products were transformed into competent cells, positive clones were screened, and mutations were verified by sequencing. Furthermore, the step of detecting the effect of the mutated transcription factor MAX on the transcriptional activity of the PD-L1 promoter to determine the specific binding motif of MAX to the PD-L1 gene promoter region includes: The effect of the mutated transcription factor MAX on the transcriptional activity of the PD-L1 promoter was detected by luciferase reporter assay, and the specific binding motif of MAX to the PD-L1 gene promoter region was determined to be: 5'-CAC[GA]TG-3'. We used Western blot to detect PD-L1 protein expression and verified the effect of motif mutation on MAX regulation of PD-L1.
[0014] Furthermore, the step of screening for DNA aptamers with high affinity for transcription factor MAX based on the specific binding motif includes: Based on the specific binding motif, DNA aptamers that bind with MAX high affinity were obtained by screening using SELEX technology.
[0015] Furthermore, the step of virtually screening small molecule compounds using a molecular docking model of the DNA binding domain of transcription factor MAX with the specific binding motif, based on the specific binding motif, includes: Small molecule compounds are virtually screened using a molecular docking model of the DNA-binding domain of transcription factor MAX with the motif, wherein the DNA-binding domain may be a bHLH domain.
[0016] Furthermore, the steps of sequentially verifying the inhibitory effect of DNA aptamers or small molecule compounds on the binding of transcription factor MAX to the PD-L1 promoter, the inhibitory effect of transcription factor MAX on the activation of the PD-L1 promoter, and the inhibitory effect on immune escape of target cancer cells include: The inhibitory effect of the DNA aptamer or small molecule compound on the binding of MAX to the PD-L1 promoter motif was verified by EMSA experiments, its inhibitory effect on MAX transcriptional activation of PD-L1 was verified by luciferase reporter assays, and its inhibitory effect on immune escape of lung adenocarcinoma cells was verified by primary CD8+ T cell killing assays.
[0017] Further, the steps of determining the dissociation constant of DNA aptamers or small molecule compounds with transcription factor MAX through SPR experiments, screening for high-affinity molecules with dissociation constants less than a first threshold, and further detecting the inhibition rate of high-affinity molecules on the binding of specific binding motifs through EMSA experiments to screen for highly active inhibitors with inhibition rates greater than a second threshold include: The dissociation constant Kd was determined by SPR experiment, and high-affinity molecules with Kd < 100 nM were screened. Furthermore, the inhibition rate of the high-affinity molecules on the binding of MAX to the PD-L1 promoter-specific binding motif was detected by EMSA experiment, and high-activity inhibitors with inhibition rate > 80% were screened.
[0018] Furthermore, the step of modifying the validated DNA aptamers with cholesterol includes: Cholesterol was modified to the 3' end of the DNA aptamer via a disulfide linker arm, and the disulfide bond could be specifically broken at the glutathione concentration in the tumor microenvironment. The disulfide linker arm consists of a 3'-mercaptopropyl-modified DNA aptamer and cholesterol-PEG. 2000 - Maleimide coupling.
[0019] The second approach provided in this application is: a dual-target inhibitor, wherein the dual-target inhibitor simultaneously targets the MAX-PD-L1 promoter-specific binding motif and the JAK1 kinase domain, and the dual-target inhibitor comprises: A bifunctional DNA aptamer comprising a MAX binding domain and a JAK1 binding domain connected by a flexible linker arm. The MAX binding domain is a cholesterol-modified DNA aptamer with a dissociation constant Kd < 100 nM, and the JAK1 binding domain is a high-affinity DNA aptamer screened using SELEX technology with an inhibition rate > 80%. A multi-target small molecule compound, wherein the core of the multi-target small molecule compound is a MAX inhibitor and a JAK1 inhibitory group is introduced into the structure; the multi-target small molecule compound is designed by molecular docking technology and can simultaneously bind the bHLH domain of MAX and the kinase domain of JAK1, with an IC50 < 50 nM.
[0020] The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif provided in this application has the following advantages: Through ChIP-seq and site-directed mutagenesis experiments, the core binding motif of MAX and the PD-L1 promoter, such as 5'-CAC[GA]TG-3', is identified, ensuring that the inhibitor targets only the key site of the MAX-PD-L1 interaction, avoiding off-target effects and providing a highly specific target for subsequent inhibitor design. DNA aptamers screened based on the specific binding motif, after cholesterol modification, exhibit enhanced cell permeability and significantly higher affinity than traditional antibodies. Small molecule compounds virtually screened using a molecular docking model, after optimization through hydrogen bonding and hydrophobic interactions, show improved binding affinity to MAX. After inhibitor treatment, the binding inhibition rate of MAX to the PD-L1 promoter is high, PD-L1 transcriptional activity is significantly reduced, and the killing rate of T cells against tumor cells is also improved, effectively blocking immune escape. Both the NA aptamer and the small molecule compounds undergo rigorous screening using SPR and EMSA to ensure high activity and low toxicity. Combining with PD-1 monoclonal antibodies enhances response rates and provides a combined target for immunotherapy of lung adenocarcinoma. The inhibitor prepared by this method significantly inhibits tumor immune escape by precisely targeting the MAX-PD-L1 signaling axis, exhibiting high specificity and high affinity. Attached Figure Description
[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0022] Figure 1 This is one of the flowcharts illustrating a method for preparing an inhibitor that specifically binds to the MAX-PD-L1 promoter motif, as an example. Figure 2 This is the second flowchart illustrating a method for preparing an inhibitor that specifically binds to the MAX-PD-L1 promoter motif, as an example. Figure 3 This is the third flowchart illustrating a method for preparing an inhibitor that specifically binds to the MAX-PD-L1 promoter motif, as an example. Figure 4 This is the fourth flowchart illustrating a method for preparing an inhibitor that specifically binds to the MAX-PD-L1 promoter motif, as an example. Figure 5 This is the fifth flowchart illustrating a method for preparing an inhibitor that specifically binds to the MAX-PD-L1 promoter motif, as an example. Detailed Implementation
[0023] To enable those skilled in the art to better understand the technical solutions in this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.
[0024] Example 1 Existing anti-PD-L1 inhibitors target PD-L1 protein or PD-1 receptor, which can only block the function of the synthesized PD-L1 protein and cannot continuously synthesize PD-L1 from the transcriptional source. This results in a limited response rate for anti-PD-L1 monotherapy. The fundamental reason is that tumor cells can reactivate PD-L1 expression through upstream regulatory pathways, leading to drug resistance. Existing inhibitor preparation methods do not target the PD-L1 transcriptional activation process, and therefore cannot solve the drug resistance problem.
[0025] Existing small molecule / nucleic acid drug preparation mainly focuses on protein-protein interactions, such as PD-L1-PD-1. The target design technology for transcription factor-promoter DNA interactions is relatively weak. Existing preparation methods do not have reference templates for such DNA-protein binding targets, and cannot achieve the design of highly specific inhibitors.
[0026] Furthermore, PD-L1 expression is regulated by multiple mechanisms, both exogenous and endogenous. Existing conventional inhibitors only inhibit a single protein step and cannot cope with the coordinated escape of multiple mechanisms. Inhibitors prepared using existing technologies cannot comprehensively intervene in the transcription process, resulting in limited inhibitor efficacy.
[0027] See Figure 1 As shown, this embodiment provides a method for preparing an inhibitor that specifically binds to the MAX-PD-L1 promoter motif, including the following steps: S1. Enrich the chromatin DNA fragments in the target cancer cells that directly bind to the transcription factor MAX protein, and obtain the sequence information of the chromatin DNA fragments; In step S1, chromatin DNA fragments that directly bind to MAX in target cancer cells are enriched using MAX-specific antibodies via chromatin immunoprecipitation, and the sequence information of the DNA fragments is obtained by high-throughput sequencing.
[0028] S2. Screen for MAX binding peaks located in the promoter region of the PD-L1 gene from the sequence information of the chromatin DNA fragments, and analyze the conserved sequences of the binding peaks to preliminarily determine candidate motifs. In step S2, MAX binding peaks located in the promoter region of the PD-L1 gene are screened from the ChIP-seq data. The conserved sequences of the binding peaks are analyzed to preliminarily determine candidate motifs, clarify the core binding region of MAX in the PD-L1 promoter, and narrow down the range of candidate motifs.
[0029] S3. Perform site-directed mutagenesis on candidate motifs and examine the effect of the mutated transcription factor MAX on the transcriptional activity of the PD-L1 promoter to determine the specific binding motif of MAX to the PD-L1 gene promoter region. In step S3, site-directed mutagenesis is performed on the candidate motif. S4. Based on the specific binding motif, DNA aptamers with high affinity for transcription factor MAX are screened, and small molecule compounds are virtually screened using a molecular docking model between the DNA binding domain of transcription factor MAX and the specific binding motif. In step S4, based on the specific binding motif, DNA aptamers that bind with high affinity to MAX are screened using SELEX technology. A molecular docking model of the DNA binding domain of transcription factor MAX and the specific binding motif is constructed, and small molecule compounds that can competitively bind to MAX are virtually screened from the compound library.
[0030] S5. Sequentially verify the inhibitory effect of DNA aptamers or small molecule compounds on the binding of transcription factor MAX to the PD-L1 promoter, the inhibitory effect of transcription factor MAX on the activation of the PD-L1 promoter, and the inhibitory effect on immune escape of target cancer cells. In step S6, the inhibition rate of candidate molecules on the binding of MAX to the PD-L1 promoter motif is detected by EMSA assay, the inhibitory effect of candidate molecules on MAX transcriptional activation of the PD-L1 promoter is detected by luciferase reporter assay, and the inhibitory effect of candidate molecules on immune escape of target cancer cells is detected by T cell killing assay; candidate molecules that can significantly inhibit the binding of MAX to the PD-L1 promoter, reduce PD-L1 transcriptional activity, and enhance T cell killing activity are screened out.
[0031] S6. Cholesterol modification of validated DNA aptamers to improve cell permeability, and optimization of hydrogen bonding and hydrophobic interactions of small molecule compound binding bags to improve affinity. In step S6, the validated DNA aptamers are modified with cholesterol to improve cell permeability, the binding pockets of small molecules and MAX are analyzed, and hydrogen bonding and hydrophobic interactions are optimized to improve affinity.
[0032] S7. The dissociation constant of DNA aptamers or small molecule compounds with transcription factor MAX is determined by SPR experiment. High affinity molecules with dissociation constants less than the first threshold are screened out. Furthermore, the inhibition rate of high affinity molecules on the binding of specific binding motifs is detected by EMSA experiment. Highly active inhibitors with inhibition rates greater than the second threshold are screened out.
[0033] In step S7, the dissociation constant of candidate molecules with MAX is determined by SPR (Surface Plasmon Resonance) experiments to screen for high-affinity molecules. EMSA experiments are then used to detect the inhibition rate of these high-affinity molecules on the binding of MAX to the PD-L1 promoter motif, screening for active inhibitors with high inhibition rates to obtain high-affinity and high-activity inhibitors. ChIP-seq and site-directed mutagenesis experiments are used to identify the core binding motif of MAX to the PD-L1 promoter, such as 5'-CAC[GA]TG-3', ensuring that the inhibitor targets only the key site of the MAX-PD-L1 interaction, avoiding off-target effects and providing highly specific targets for subsequent inhibitor design. DNA aptamers screened based on specific binding motifs exhibit improved cell permeability after cholesterol modification and have a dissociation constant Kd < 100 nM with MAX, showing significantly higher affinity than traditional antibodies. Small molecule compounds virtually screened using molecular docking models, after optimization of hydrogen bonding and hydrophobic interactions, show improved binding affinity to MAX, with an IC50 < 50 nM, demonstrating drug potential. After treatment with inhibitors, MAX showed a high inhibition rate of binding to the PD-L1 promoter, significantly reduced PD-L1 transcriptional activity, and enhanced T cell killing rate against tumor cells, effectively blocking immune escape. Both the NA aptamer and the small molecule compound underwent rigorous screening using SPR and EMSA to ensure high activity and low toxicity. Combination with PD-1 monoclonal antibodies can improve response rates and provide a combined target for immunotherapy of lung adenocarcinoma.
[0034] The inhibitor prepared by this method significantly inhibits tumor immune escape by precisely targeting the MAX-PD-L1 signaling axis, and has high specificity, high affinity and good prospects for clinical translation.
[0035] This embodiment provides further technical solutions, please refer to... Figure 2 As shown, the step of enriching chromatin DNA fragments that directly bind to the transcription factor MAX protein in target cancer cells and obtaining the sequence information of these chromatin DNA fragments includes: S11. Chromatin DNA fragments that directly bind to the transcription factor MAX protein in lung adenocarcinoma cells are enriched using chromatin immunoprecipitation technology, and the sequence information of the chromatin DNA fragments is obtained by high-throughput sequencing.
[0036] It should be noted that the immunoprecipitation technique can efficiently enrich chromatin DNA fragments that directly bind to MAX, with an enrichment efficiency of 0.1%-1% of the input DNA. Combined with high-throughput sequencing and bioinformatics analysis, the binding peak of MAX can be identified across the entire genome at a resolution down to the base level, and the binding site of MAX in the PD-L1 promoter region can be clearly identified, thus achieving precise enrichment and identification of the binding site of MAX to chromatin DNA.
[0037] This embodiment provides a further technical solution, wherein the lung adenocarcinoma cells include A549 and H1975.
[0038] It should be noted that A549 cells are a classic lung adenocarcinoma cell line carrying wild-type KRAS and EGFR mutations, widely used in basic lung cancer research, and their biological behavior is highly consistent with clinical lung adenocarcinoma. H1975 cells carry the EGFR L858R / T790M double mutation and are a common EGFR-TKI resistance model in clinical practice.
[0039] This embodiment provides further technical solutions, please refer to... Figure 3 As shown, the steps of screening for MAX binding peaks located in the PD-L1 gene promoter region from the sequence information of the autochromatin DNA fragments, and analyzing the conserved sequences of the binding peaks to preliminarily determine candidate motifs include: S21. The sequence information of the autochromatin DNA fragment was compared with that of the human genome hg38. The MAX binding peak located in the promoter region of the PD-L1 gene was screened using IGV software for visualization analysis. The conserved sequence of the binding peak region was analyzed by MEME Suite to preliminarily determine the candidate motif.
[0040] In step S21, the raw sequences of DNA fragments enriched by chromatin immunoprecipitation are obtained through high-throughput sequencing. These sequences are then aligned with the human genome reference sequence using Bowtie2 software to generate a SAM format file. The SAM file is converted to BAM format and sorted using SAMtools, removing PCR repetitive sequences to obtain high-quality alignment results. The sorted BAM file is imported into IGV (Integrative Genomics Viewer, a genome data visualization tool) to locate the promoter region of the PD-L1 gene. Through visualization analysis, regions with significantly higher coverage than the background are selected, and the genomic coordinates of the binding peaks are recorded. The DNA sequences of the MAX binding peaks in the PD-L1 promoter region selected from IGV are extracted. Conserved motifs in the binding peak regions are analyzed, and the predicted conserved motifs are compared with a database of known transcription factor binding motifs to verify their consistency with known MAX binding motifs, ultimately determining candidate motifs.
[0041] This example provides a further technical solution, wherein the PD-L1 gene promoter region is 2kb upstream to 500bp downstream of the transcription start site.
[0042] It should be noted that the PD-L1 gene promoter region is defined as 2kb upstream to 500bp downstream of the transcription start site. This region covers the key regulatory regions of PD-L1 gene transcription initiation, such as the core promoter and proximal enhancer. Combined with ChIP-seq data, the specific binding peak of MAX can be accurately identified.
[0043] Example 2 Based on Embodiment 1, this embodiment provides further technical solutions.
[0044] This embodiment provides further technical solutions, please refer to... Figure 4 As shown, the step of performing site-directed mutagenesis on the candidate motif includes: S311. For candidate motifs in the PD-L1 promoter region, design complementary primers that cover the mutation region, with the mutation site located in the middle of the primer and restriction sites introduced at both ends. In step S311, a pair of complementary mutant primers are designed for candidate motifs in the PD-L1 promoter region, such as 5'-CACGTG-3'. The primers are 25-30 bp in length, with the mutation site (e.g., mutating 5'-CACGTG-3' to 5'-GAGCTG-3') located in the middle of the primers, and restriction enzyme sites matching the template plasmid introduced at both ends.
[0045] S312. Using a plasmid containing the PD-L1 promoter as a template, perform PCR amplification with high-fidelity DNA polymerase to obtain a plasmid containing the mutation site. In step S312, PCR amplification is performed using a plasmid containing the wild-type PD-L1 promoter as a template and high-fidelity DNA polymerase.
[0046] S313. Digest methylated template DNA with Dpnl restriction endonuclease and retain unmethylated mutant plasmids; In step S313, DpnI restriction endonuclease is added to the PCR product. DpnI specifically recognizes and cleaves the methylated template plasmid, while the mutant plasmid amplified by PCR is unmethylated and therefore retained.
[0047] S314. Transform the digestion products into competent cells, screen for positive clones, and sequence them to verify the mutations.
[0048] In step S314, mutant plasmids with correct sequences are obtained through transformation and screening, thereby improving the mutation success rate.
[0049] This embodiment provides further technical solutions, please refer to... Figure 5 As shown, the step of detecting the effect of the mutated transcription factor MAX on the transcriptional activity of the PD-L1 promoter to determine the specific binding motif of MAX to the PD-L1 gene promoter region includes: S321. The effect of the mutated transcription factor MAX on the transcriptional activity of the PD-L1 promoter was detected by luciferase reporter assay, and the specific binding motif of MAX to the PD-L1 gene promoter region was determined to be: 5'-CAC[GA]TG-3'. In step S321, using the wild-type PD-L1 promoter as a template, key bases in the motif were inserted into the pGL3-Basic vector through site-directed mutagenesis to construct a mutant reporter plasmid; the wild-type reporter plasmid was retained as a control. A549 cells or H1975 cells were seeded in 24-well plates. When the cell confluence reached 70%-80%, they were transfected in groups. Forty-eight hours after transfection, cells were lysed using a dual-luciferase reporter gene detection system to measure the activities of firefly luciferase and Renilla luciferase, and the relative luciferase activities were calculated.
[0050] S322. Western blot was used to detect PD-L1 protein expression and verify the effect of motif mutation on MAX regulation of PD-L1.
[0051] In step S322, after A549 cells were grouped and transfected, total protein was extracted, protein concentration was measured, and it was verified that after the motif mutation, the regulatory effect of MAX on PD-L1 protein expression disappeared, further confirming that the 5'-CAC[GA]TG-3' motif is the key target of MAX in regulating PD-L1.
[0052] This embodiment provides a further technical solution, wherein the step of screening for DNA aptamers with high affinity for transcription factor MAX based on the specific binding motif includes: Based on the specific binding motif, DNA aptamers that bind with MAX high affinity were obtained by screening using SELEX technology.
[0053] It should be noted that through 8-12 rounds of SELEX screening, DNA aptamers that bind with high affinity to MAX are obtained. These high-affinity aptamers can serve as inhibitor candidates for the MAX-PD-L1 signaling axis, inhibiting PD-L1 transcription by blocking the binding of MAX to the PD-L1 promoter.
[0054] This embodiment provides a further technical solution. Based on the specific binding motif, the steps of virtually screening small molecule compounds using a molecular docking model of the DNA binding domain of transcription factor MAX and the specific binding motif include: Small molecule compounds are virtually screened using a molecular docking model of the DNA-binding domain of transcription factor MAX with the motif, wherein the DNA-binding domain may be a bHLH domain.
[0055] It should be noted that virtual screening can quickly identify small molecule compounds that bind with high affinity to the bHLH domain of MAX from the compound library, thereby improving screening efficiency.
[0056] This embodiment provides a further technical solution, wherein the steps of sequentially verifying the inhibitory effect of DNA aptamers or small molecule compounds on the binding of transcription factor MAX to the PD-L1 promoter, the inhibitory effect of transcription factor MAX on the activation of the PD-L1 promoter, and the inhibitory effect on immune escape of target cancer cells include: The inhibitory effect of the DNA aptamer or small molecule compound on the binding of MAX to the PD-L1 promoter motif was verified by EMSA experiments, its inhibitory effect on MAX transcriptional activation of PD-L1 was verified by luciferase reporter assays, and its inhibitory effect on immune escape of lung adenocarcinoma cells was verified by primary CD8+ T cell killing assays.
[0057] It should be noted that the inhibitors can enhance the killing effect of T cells on tumor cells. The inhibitory effect of DNA aptamers / small molecule compounds on the MAX-PD-L1 signaling axis was systematically verified by EMSA, luciferase reporter assay and T cell killing assay.
[0058] This embodiment provides a further technical solution. The steps of determining the dissociation constant of DNA aptamers or small molecule compounds with transcription factor MAX through SPR experiments, screening for high-affinity molecules with dissociation constants less than a first threshold, and further detecting the inhibition rate of high-affinity molecules on the binding of specific binding motifs through EMSA experiments to screen for highly active inhibitors with inhibition rates greater than a second threshold include: The dissociation constant Kd was determined by SPR experiment, and high-affinity molecules with Kd < 100 nM were screened. Furthermore, the inhibition rate of the high-affinity molecules on the binding of MAX to the PD-L1 promoter-specific binding motif was detected by EMSA experiment, and high-activity inhibitors with inhibition rate > 80% were screened.
[0059] It should be noted that the precise binding affinity between DNA aptamers / small molecule compounds and MAX was obtained, high-affinity candidate molecules were screened, and the inhibition rate of high-affinity molecules on the binding of MAX to the PD-L1 promoter motif was verified by EMSA experiments. This clarified the inhibitory ability of high-affinity molecules on MAX-DNA binding, and highly active inhibitors with inhibition rates >80% were screened. The complex band significantly weakened with increasing inhibitor concentration. Validation by SPR and EMSA ensured that the screened inhibitors had clear molecular targeting.
[0060] This embodiment provides a further technical solution, wherein the step of modifying the verified DNA aptamer with cholesterol includes: Cholesterol was modified to the 3' end of the DNA aptamer via a disulfide linker arm, and the disulfide bond could be specifically broken at the glutathione concentration in the tumor microenvironment. The disulfide linker arm consists of a 3'-mercaptopropyl-modified DNA aptamer and cholesterol-PEG. 2000 - Maleimide coupling.
[0061] It should be noted that cholesterol modification can enhance the cell permeability of DNA aptamers, but traditional cholesterol-modified aptamers are easily taken up non-specifically in normal tissues, leading to off-target toxicity. At the same time, after entering the cell, the aptamers are difficult to release in the tumor microenvironment, affecting their binding efficiency with MAX.
[0062] Therefore, this embodiment solves the off-target and release problems of traditional cholesterol-modified aptamers by tumor microenvironment-responsive modification, significantly improving the targeting and activity of the inhibitor, and providing a better technical solution for the precise inhibition of the MAX-PD-L1 signaling axis.
[0063] Specifically, cholesterol and DNA aptamers are linked by disulfide bonds that can be specifically broken by high concentrations of GSH in the tumor microenvironment. Cholesterol is modified at the 3' end of the DNA aptamer to avoid interfering with the binding of the aptamer to MAX. The purity of the modified aptamer is detected by HPLC, and the particle size change at different GSH concentrations is detected by dynamic light scattering.
[0064] Cholesterol modification can improve the cell membrane permeability of DNA aptamers; PEG 2000 The chain extends the circulating half-life, specifically cleaves disulfide bonds in the high GSH concentration of the tumor microenvironment, releases free DNA aptamers, avoids toxicity to normal tissues, and maintains stable binding affinity between the modified DNA aptamer and MAX. This is achieved through cholesterol-PEGylation. 2000 - Maleimide was used to prepare tumor microenvironment-responsive DNA aptamers, providing a guarantee for targeted therapy along the MAX-PD-L1 signaling axis.
[0065] Example 3 Based on the above embodiments, the technical problem solved by this implementation is that traditional cholesterol-modified DNA aptamers only enhance permeability through membrane insertion, but lack a tumor cell-specific recognition mechanism. They easily bind to normal lung epithelial cells, leading to off-target effects, which reduce the effective drug concentration at the tumor site and increase the risk of damage to normal tissues, resulting in a narrow therapeutic window. If the targeting peptide is directly coupled to the aptamer, the targeting peptide and the cholesterol-modified / aptamer functional domain, i.e., the MAX binding region, easily form steric hindrance, hindering cholesterol insertion into the cell membrane. This impairs membrane permeability or causes the aptamer to bind to the MAX protein, leading to loss of inhibitory activity. In addition, existing disulfide-modified aptamers often present a contradiction between easy breakage in the normal environment and difficulty in release in the tumor environment. The former leads to premature degradation of the aptamer in the bloodstream, while the latter prevents effective release of active molecules, both affecting the inhibitory effect.
[0066] To address this technical challenge, this embodiment specifically provides a method for preparing an inhibitor that specifically targets the MAX-PD-L1 promoter binding motif. Specifically, this method involves a procedure for coupling an EGFR-targeting peptide to the 5' end of a cholesterol-modified DNA aptamer. T1: Synthesize MAX-binding DNA aptamers modified with 5' amino group and 3' thiol group, purify them to a purity of ≥95% by reversed-phase high-performance liquid chromatography (HPLC), and freeze-dry for later use; T2: An EGFR-targeting peptide with the amino acid sequence YHWYGYTPQNVI was synthesized in a solid phase. After introducing a carboxyl group modification at the C-terminus, the carboxyl group was activated by 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and covalently linked to the 5' amino group of the aptamer through a flexible linker composed of 3-5 glycine-serine repeating units. T3: Transforms cholesterol via PEG 2000 - The dithiopyridine reagent forms a disulfide bond with the 3' end thiol group of the aptamer, and the reaction conditions are incubation at 37°C for 2 hours. T4: Dialyze for 24 hours using a dialysis bag with a molecular weight cutoff of 3 kDa, changing the medium every 8 hours to remove small molecule impurities. Then, purify the product a second time by reversed-phase HPLC. Mass spectrometry verifies that the molecular weight error of the product is ≤0.1%, and agarose gel electrophoresis confirms the integrity of the product. T5: Test the disulfide bond stability of the product to ensure that its breakage rate is ≥90% within 1 hour in a 10mM glutathione solution.
[0067] The principle behind steps T1-T5 is that cholesterol is coupled to the 3' end of the aptamer via a disulfide bond linker arm, allowing it to insert into the phospholipid bilayer of the cell membrane and promoting aptamer transmembrane entry into the cell. The disulfide bond breaks under the high glutathione concentration in the tumor microenvironment, releasing the active aptamer to bind to MAX protein. Lung adenocarcinoma cells highly express the EGFR receptor; the EGFR-targeting peptide can specifically bind to the extracellular domain of EGFR, guiding the aptamer complex to preferentially accumulate on the tumor cell surface, reducing off-target effects. Using Gly-Ser repeat units as linkers, their flexible structure avoids spatial interference between the targeting peptide and the cholesterol / aptamer functional domain, ensuring that the aptamer maintains its binding activity to MAX after entering the cell.
[0068] That is, based on Example 2, this solution solves the off-target problem of cholesterol-modifying aptamers by optimizing the targeting peptide and flexible linker, and significantly improves tumor targeting and therapeutic window.
[0069] Example 4 This embodiment provides a dual-target inhibitor, prepared using an inhibitor preparation method targeting the MAX-PD-L1 promoter-specific binding motif as described in Embodiment 1 or Example 2 above. The dual-target inhibitor simultaneously targets both the MAX-PD-L1 promoter-specific binding motif (5'-CAC[GA]TG-3') and the JAK1 kinase domain. The dual-target inhibitor comprises: A bifunctional DNA aptamer comprising a MAX binding domain and a JAK1 binding domain connected by a flexible linker arm. The MAX binding domain is a cholesterol-modified DNA aptamer with a dissociation constant Kd < 100 nM, and the JAK1 binding domain is a high-affinity DNA aptamer screened using SELEX technology with an inhibition rate > 80%. A multi-target small molecule compound, wherein the core of the multi-target small molecule compound is a MAX inhibitor and a JAK1 inhibitory group is introduced into the structure; the multi-target small molecule compound is designed by molecular docking technology and can simultaneously bind the bHLH domain of MAX and the kinase domain of JAK1, with an IC50 < 50 nM.
[0070] It should be noted that, through ChIP-seq and site-directed mutagenesis experiments, the core binding motifs of MAX and the PD-L1 promoter, such as 5'-CAC[GA]TG-3', were identified. This ensures that the inhibitor targets only the key sites of the MAX-PD-L1 interaction, avoiding off-target effects and providing highly specific targets for subsequent inhibitor design. DNA aptamers screened based on specific binding motifs showed improved cell permeability and significantly higher affinity than traditional antibodies after cholesterol modification. Small molecule compounds virtually screened using molecular docking models, after optimization through hydrogen bonding and hydrophobic interactions, exhibited enhanced binding affinity to MAX. Inhibitor treatment resulted in high inhibition rates of MAX binding to the PD-L1 promoter, significantly reduced PD-L1 transcriptional activity, and increased T cell killing rate against tumor cells, effectively blocking immune escape.
[0071] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0072] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of this patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims.
Claims
1. A method for preparing an inhibitor targeting a MAX-PD-L1 promoter-specific binding motif, characterized in that, Including the following steps: Enrich chromatin DNA fragments in target cancer cells that directly bind to the transcription factor MAX protein, and obtain the sequence information of these chromatin DNA fragments; MAX binding peaks located in the promoter region of the PD-L1 gene were screened from the sequence information of chromatin DNA fragments, and the conserved sequences of the binding peaks were analyzed to preliminarily determine candidate motifs. Candidate motifs were subjected to site-directed mutagenesis, and the effect of the mutated transcription factor MAX on the transcriptional activity of the PD-L1 promoter was examined to determine the specific binding motif of MAX to the PD-L1 gene promoter region. Based on the specific binding motif, DNA aptamers with high affinity for transcription factor MAX were screened, and small molecule compounds were virtually screened using a molecular docking model between the DNA binding domain of transcription factor MAX and the specific binding motif. The inhibitory effects of DNA aptamers or small molecule compounds on the binding of transcription factor MAX to the PD-L1 promoter, the inhibitory effect of transcription factor MAX on the activation of the PD-L1 promoter, and the inhibitory effect on immune escape of target cancer cells were verified sequentially. Cholesterol modification was performed on the validated DNA aptamers to improve cell permeability, and hydrogen bonding and hydrophobic interactions were optimized in the binding pockets of small molecule compounds to improve affinity. The dissociation constants of DNA aptamers or small molecule compounds with transcription factor MAX were determined by SPR assay. High-affinity molecules with dissociation constants less than the first threshold were screened out. Furthermore, the inhibition rate of high-affinity molecules on the binding of specific binding motifs was detected by EMSA assay, and highly active inhibitors with inhibition rates greater than the second threshold were screened out.
2. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 1, characterized in that, The step of enriching chromatin DNA fragments in target cancer cells that directly bind to the transcription factor MAX protein and obtaining the sequence information of these chromatin DNA fragments includes: Chromatin DNA fragments that directly bind to the transcription factor MAX protein in lung adenocarcinoma cells were enriched using chromatin immunoprecipitation technology, and the sequence information of the chromatin DNA fragments was obtained by high-throughput sequencing.
3. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 2, characterized in that, The lung adenocarcinoma cells include A549 and H1975.
4. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 1, characterized in that, The steps of screening for MAX binding peaks located in the PD-L1 gene promoter region from the sequence information of the autochromatin DNA fragments, and analyzing the conserved sequences of the binding peaks to preliminarily determine candidate motifs include: The sequence information of the autochromatin DNA fragment was compared with that of the human genome hg38. Visual analysis was performed using IGV software to screen out the MAX binding peak located in the promoter region of the PD-L1 gene. The conserved sequence of the binding peak region was analyzed using MEME Suite to preliminarily determine candidate motifs.
5. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 4, characterized in that, The PD-L1 gene promoter region extends from 2 kb upstream to 500 bp downstream of the transcription start site.
6. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 1, characterized in that, The step of performing site-directed mutagenesis on the candidate motif includes: For candidate motifs in the PD-L1 promoter region, complementary primers covering the mutant region were designed, with the mutant site located in the middle of the primer and restriction sites introduced at both ends. Using a plasmid containing the PD-L1 promoter as a template, high-fidelity DNA polymerase was used for PCR amplification to obtain a plasmid containing the mutation site. Methylated template DNA was digested with Dpnl restriction endonuclease, while unmethylated mutant plasmids were retained; The digestion products were transformed into competent cells, positive clones were screened, and mutations were verified by sequencing.
7. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 1, characterized in that, The steps for detecting the effect of the mutated transcription factor MAX on the transcriptional activity of the PD-L1 promoter to determine the specific binding motif of MAX to the PD-L1 gene promoter region include: The effect of the mutated transcription factor MAX on the transcriptional activity of the PD-L1 promoter was detected by luciferase reporter assay, and the specific binding motif of MAX to the PD-L1 gene promoter region was determined to be: 5'-CAC[GA]TG-3'. We used Western blot to detect PD-L1 protein expression and verified the effect of motif mutation on MAX regulation of PD-L1.
8. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 1, characterized in that, The step of screening for DNA aptamers with high affinity for transcription factor MAX based on the specific binding motif includes: Based on the specific binding motif, DNA aptamers that bind with MAX high affinity were obtained by screening using SELEX technology.
9. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 1, characterized in that, The steps of virtually screening small molecule compounds based on the specific binding motif using a molecular docking model of the DNA binding domain of transcription factor MAX and the specific binding motif include: Small molecule compounds are virtually screened using a molecular docking model of the DNA-binding domain of transcription factor MAX with the motif, wherein the DNA-binding domain may be a bHLH domain.
10. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 1, characterized in that, The steps of sequentially verifying the inhibitory effect of DNA aptamers or small molecule compounds on the binding of transcription factor MAX to the PD-L1 promoter, the inhibitory effect of transcription factor MAX on the activation of the PD-L1 promoter, and the inhibitory effect on immune escape of target cancer cells include: The inhibitory effect of the DNA aptamer or small molecule compound on the binding of MAX to the PD-L1 promoter motif was verified by EMSA experiments, its inhibitory effect on MAX transcriptional activation of PD-L1 was verified by luciferase reporter assays, and its inhibitory effect on immune escape of lung adenocarcinoma cells was verified by primary CD8+ T cell killing assays.
11. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 1, characterized in that, The steps of determining the dissociation constant of DNA aptamers or small molecule compounds with transcription factor MAX through SPR experiments, screening for high-affinity molecules with dissociation constants less than a first threshold, and further detecting the inhibition rate of high-affinity molecules on the binding of specific binding motifs through EMSA experiments to screen for highly active inhibitors with inhibition rates greater than a second threshold include: The dissociation constant Kd was determined by SPR experiment, and high-affinity molecules with Kd < 100 nM were screened. Furthermore, the inhibition rate of the high-affinity molecules on the binding of MAX to the PD-L1 promoter-specific binding motif was detected by EMSA experiment, and high-activity inhibitors with inhibition rate > 80% were screened.
12. The method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif according to claim 1, characterized in that, The step of modifying the validated DNA aptamers with cholesterol includes: Cholesterol was modified to the 3' end of the DNA aptamer via a disulfide linker arm, and the disulfide bond could be specifically broken at the glutathione concentration in the tumor microenvironment. The disulfide linker arm consists of a 3'-mercaptopropyl-modified DNA aptamer and cholesterol-PEG. 2000 - Maleimide coupling.
13. A dual-target inhibitor, characterized in that, The inhibitor is prepared by the method for preparing an inhibitor targeting the MAX-PD-L1 promoter-specific binding motif as described in any one of claims 1 to 12, wherein the dual-target inhibitor simultaneously targets both the MAX-PD-L1 promoter-specific binding motif and the JAK1 kinase domain, and the dual-target inhibitor comprises: A bifunctional DNA aptamer comprising a MAX binding domain and a JAK1 binding domain connected by a flexible linker arm. The MAX binding domain is a cholesterol-modified DNA aptamer with a dissociation constant Kd < 100 nM, and the JAK1 binding domain is a high-affinity DNA aptamer screened using SELEX technology with an inhibition rate > 80%. A multi-target small molecule compound, wherein the core of the multi-target small molecule compound is a MAX inhibitor and a JAK1 inhibitory group is introduced into the structure; the multi-target small molecule compound is designed by molecular docking technology and can simultaneously bind the bHLH domain of MAX and the kinase domain of JAK1, with an IC50 < 50 nM.