New medicine research and development method for treating esophageal squamous carcinoma

Abstract

The invention discloses a research and development method of a new drug for treating esophageal squamous cell carcinoma, and belongs to the technical field of antitumor drugs. The structural formula of the compound A6 is as shown in the formula I. The compound A6 can be prepared into a medicine to play the anti-esophageal squamous carcinoma roles of promoting apoptosis of esophageal squamous carcinoma cells, inhibiting proliferation, invasion or migration of esophageal squamous carcinoma cells and the like. The medicine prepared according to the compound A6 is the first esophageal squamous cell carcinoma phase separation targeted medicine and is also the first early-stage esophageal squamous cell carcinoma targeted medicine, the market blank is filled, and the compound A6 has a huge cancer treatment prospect.

Description

Technical Field

[0001] This invention belongs to the field of antitumor drug technology, specifically relating to a new drug development method for the treatment of esophageal squamous cell carcinoma. Background Technology

[0002] Traditional targeted drugs rely on the binding pockets of ordered protein structures, while phase-separated targeted drugs can target disordered regions of proteins, achieving functional intervention by modulating weak intramolecular interactions. The development of phase-separated drugs has significantly expanded the range of druggable targets, transforming proteins traditionally considered "undruggable" into "drug-prone" ones, and overcoming the drug resistance bottlenecks of existing targets. Therefore, phase-separated targeted drugs not only possess unique advantages but can also complement traditional targeted drugs. Figure 1 ).

[0003] Esophageal cancer has a persistently high incidence and mortality rate. Due to the lack of obvious early symptoms, most patients are diagnosed at an advanced stage. Current treatments for esophageal squamous cell carcinoma (ESCC) primarily rely on traditional radiotherapy and chemotherapy. Existing targeted therapies (EGFR inhibitors, VEGF inhibitors, PD-1 / PD-L1 inhibitors) are broad-spectrum drugs targeting tumors, lacking specific targets and intervention methods for ESCC. Furthermore, these targeted drugs can only be used as second-line treatment for advanced stages, with limited efficacy and significant side effects. Specific targeted drugs for early and mid-stage ESCC are completely absent. The problems of radiotherapy and chemotherapy resistance and the lack of effective targeted drugs have become core bottlenecks that the pharmaceutical industry urgently needs to address. Therefore, the development of new anti-ESCC drugs is urgently needed. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides the application of compound A6 in the preparation of drugs for treating esophageal squamous cell carcinoma, providing a new target for treating esophageal squamous cell carcinoma, demonstrating significant innovation and potential clinical value in the field of anticancer drug development.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is to provide the application of compound A6 in the preparation of a drug for treating esophageal squamous cell carcinoma. The structural formula of compound A6 is shown in Formula I. .

[0006] Based on the above technical solution, the present invention can be further improved as follows.

[0007] Furthermore, the drug contains compound A6 and / or its pharmaceutically acceptable salt.

[0008] Furthermore, the drug is in the form of tablets, capsules, powders, pills, granules, gels, injections, or emulsions.

[0009] Furthermore, esophageal squamous cell carcinoma is caused by KYSE150 cells or TE-1 cells.

[0010] Furthermore, the anti-esophageal squamous cell carcinoma treatment includes promoting apoptosis of esophageal squamous cell carcinoma cells and inhibiting the proliferation, invasion, and / or migration of esophageal squamous cell carcinoma cells.

[0011] The beneficial effects of this invention are as follows: The phase separation targeted drug (A6) obtained based on the artificial intelligence phase separation drug screening platform is the world's first phase separation targeted drug for esophageal squamous cell carcinoma, and also the world's first targeted drug for early-stage esophageal squamous cell carcinoma. Compound A6 exhibits excellent selective killing activity, significantly inhibiting the proliferation, migration, and invasion of esophageal squamous cell carcinoma cell lines in in vitro experiments, inducing apoptosis, and showing low toxicity to normal esophageal epithelial cells, demonstrating excellent tumor specificity. Attached Figure Description

[0012] Figure 1 TFAP2β was identified as a key transcription factor that was significantly downregulated in EESCC; (A) shows the ATAC-seq experimental procedure; (B) shows a schematic diagram of early ESCC patient sample collection and a representative H&E staining image, with a scale bar of 200 μm. Figure 2 TFAP2β was identified as a key transcription factor significantly downregulated in EESCC. (A) Principal component analysis (PCA) of chromatin accessibility at P, Tm, and Tsm tissues; (B, C) Volcano plots of differentially accessible peaks (DA peaks, ATAC-seq) between Tm and P (B) and differentially expressed genes (DE genes, RNA-seq) (C), respectively; (D, E) overlap (D) and correlation analysis (E) between differentially accessible peaks and differentially expressed genes, respectively; (F) Venn diagram: overlap between motif-enriched transcription factors and upregulated / downregulated transcription factors in Tm vs. P (red) and Tsm vs. P (blue) comparisons; (G) Heatmap of representative transcription factors (TFAP2β, ZIC2, NFIX, ID4); (H) Kaplan-Meier survival analysis of TFAP2β expression levels in the ESCC (TCGA cohort). Figure 3To investigate the effects of TFAP2β on promoting apoptosis and inhibiting the proliferation, migration, and invasion of ESCC cells, the study included: (A) Immunohistochemical staining (IHC) of TFAP2β in paired P, early, and late ESCC tissues from patient #1 (scale bar: 40 μm); (B, C) Western blot analysis of TFAP2β in samples (B) and cell line (C) from patients #1-#6 (n=6), respectively; (D, E) Clonogenesis experiments performed in KYSE150 and TE-1 cells after overexpression of TFAP2β, respectively; (FI) CCK-8 assay: overexpression of empty vector / TFAP2β in KYSE150 (F) and TE-1 (G) cells; and treatment with si-NC and si-TFAP2β in TE-1 (H) and HET-1A (I) cells, respectively; (JL) TFAP2β assays performed in different cell lines. Flow cytometry (FACS) after RNA interference (RNAi) or overexpression (OP) (J), scratch healing assay (K), and Transwell assay (L); Figure 4 To investigate how TFAP2β promotes apoptosis and inhibits the proliferation, migration, and invasion of ESCC cells; (A) is a schematic diagram of a xenograft tumor and metastatic mouse model, using KYSE150 cells expressing GFP or GFP-TFAP2β; (B, C) are the subcutaneous xenograft tumor volume (B) and growth curve (C) respectively (n=12); (DF) are the metastasis imaging results (D) and representative liver (E) and lung tissue (F) images, scale bar: 1 cm; (G, H) are the quantitative analysis of the number of metastatic nodules in the liver (G) and lung (H) respectively; Figure 5 TFAP2β exhibits aggregation (phase separation) properties both intranuclearly and in vitro. (A) shows immunostaining of TFAP2β in P and Tm tissues, with arrows indicating TFAP2β puncta (scale bar: 5 μm); (BD) shows quantitative analysis of the proportion of puncta-positive cells (B), the number of puncta (C), and the TFAP2β signal intensity (D); (EG) shows immunostaining of TFAP2β in HET-1A, TE-1, and KYSE150 cells (E), and quantitative analysis of the number (F) and intensity (G) of TFAP2β puncta (scale bar: 5 μm); (H, I) shows the FRAP experiment (n=5) of GFP-TFAP2β intranuclear condensates in KYSE150 cells (scale bar: 5 μm); (J) shows the reversible changes of GFP-TFAP2β condensates under different pH conditions: dissolution at pH 10 and reformation at pH 7 (scale bar: 5 μm). Figure 6TFAP2β exhibits aggregation (phase separation) properties both in the cell nucleus and in vitro. (A) is a CLEM image of GFP-TFAP2β aggregates in KYSE150 cells, scale bar: left 5 μm, center 0.5 μm, right 0.25 μm; (B) is the droplet radius distribution of GFP-TFAP2β determined by dynamic light scattering (DLS); (C, D) are the experimental results of droplet formation of mCherry-labeled (C) and GFP-labeled (D) TFAP2β in vitro, scale bar: 5 μm; (E, F) are the fusion phenomena of mCherry-TFAP2β (E) and GFP-TFAP2β (F) droplets, scale bar: 5 μm; (GJ) is the FRAP experiment of mCherry-TFAP2β (G, I) and GFP-TFAP2β (H, J) droplets, showing the fusion before bleaching (-3 s) and during bleaching (0 t). min) and after bleaching (30 min), scale bar: 5 μm; Figure 7This study aims to illustrate the role of TFAP2β in ESCC cell proliferation, apoptosis, and migration through its aggregation (phase separation) properties. (A) shows a structural schematic of TFAP2β and its mutants. (B) shows differential interference contrast (DIC) images of His-TFAP2β, His-ΔIDR1, His-ΔIDR2, His-Mut_IDR2, His-ΔIDR2-FUS, and His-Mut_IDR2-FUS protein droplets, scale bar: 10 μm. (C) shows imaging of GFP-TFAP2β, GFP-TFAP2β-ΔIDR2, GFP-TFAP2β-Mut_IDR2, GFP-TFAP2β-ΔIDR2-FUS, and GFP-TFAP2β-Mut_IDR2-FUS (green) in KYSE150 cells, with DAPI (blue), scale bar: 10 μm. μm; (DG) represents the effect of overexpression of GFP, GFP-TFAP2β, GFP-TFAP2β-ΔIDR2, GFP-TFAP2β-Mut_IDR2, GFP-TFAP2β-ΔIDR2-FUS, or GFP-TFAP2β-Mut_IDR2-FUS on cell phenotype: cell proliferation was detected by CCK-8 assay (D), apoptosis by flow cytometry (FACS) (E), and cell migration (F) and invasion (G) by Transwell assay; (HK) represents the effect of expression of TFAP2β-NS, TFAP2β-ΔIDR2-NS, TFAP2β-Mut_IDR2-NS, TFAP2β-ΔIDR2-FUS-NS, or TFAP2β-Mut_IDR2-FUS-NS on cell proliferation (CCK-8) (H), apoptosis (FACS) (I), and cell migration (J) and invasion (K) (Transwell) in TFAP2β RNA interference cells; Figure 8 To illustrate the role of TFAP2β in ESCC cell proliferation, apoptosis, and migration through its aggregation (phase separation) properties; (A) is a schematic diagram of subcutaneous and tail vein xenograft models; (BD) shows the tumor volume (B), growth curve (C), and immunofluorescence staining results (D) of xenografts overexpressing different TFAP2β mutants, with arrows indicating intranuclear spots, scale bar: 10 μm; (E) shows the imaging results of tail vein transfection mice overexpressing different TFAP2β mutants; (F, G) are representative liver (F) and lung tissue (G) images, with circles indicating metastatic lesions, scale bar: 10 mm; (H, I) is a statistical analysis of the number of metastatic nodules in the liver (H) and lung (I); Figure 9To investigate the role of TFAP2β condensates in inhibiting ZNF131 transcription and recruiting other transcription factors, the study included: (A) a Venn diagram showing the overlap of genes regulated by TFAP2β; (B) a genomic trajectory map of the ZNF131 gene locus in KYSE150 cells expressing TFAP2β or its mutants, based on ChIP-seq and RNA-seq data; (C) FISH and smFISH showing the colocalization of GFP-TFAP2β with the ZNF131 promoter focus, scale bar: 5 μm; and (DF) ChIP-qPCR (D), RT-qPCR (E), and luciferase reporter gene assays (F) under conditions of overexpression of TFAP2β and its mutants. Figure 10 The study aimed to inhibit ZNF131 transcription and recruit other transcription factors through TFAP2β condensate expression. (AD) shows that ZNF131 overexpression reversed the effects of TFAP2β overexpression on cell proliferation (A), apoptosis (B), migration (C), and invasion (D). (EH) shows the effects of si-NC, si-TFAP2β, si-ZNF131, or si-TFAP2β+si-ZNF131 treatment on cell proliferation (E), apoptosis (F), and cell migration (G) and invasion (H) using a Transwell assay, respectively. (I) shows the effect of ZNF131 overexpression on the empty vector or YAP1 in KYSE150 cells. (J) RT-qPCR quantitative analysis of mRNA level; (K) ChIP-qPCR analysis of ZNF131 promoter activity after overexpression of empty vector or YAP1; (J) ChIP-qPCR analysis of YAP1 enrichment in ZNF131 promoter region under overexpression of GFP-TFAP2β, GFP-TFAP2β-Mut_IDR2 or GFP-TFAP2β-Mut_IDR2-FUS. Figure 11 To investigate the role of TFAP2β condensates in inhibiting ZNF131 transcription and recruiting other transcription factors; (A) is a schematic diagram of the droplet pelleting assay; (B) shows the analysis of NFIX and ID4 incorporation into GFP-TFAP2β or GFP-FUS condensates using ESCC cell lysates, with results expressed as Western blot; (C, D) show colocalization between TFAP2β and NFIX spots as displayed by live-cell imaging; scale bar: 5 μm; (E) shows the ChIP-qPCR analysis of NFIX enrichment in the Meis1 enhancer region under conditions of overexpression of GFP-TFAP2β, GFP-TFAP2β-Mut_IDR2, or GFP-TFAP2β-Mut_IDR2-FUS. Figure 12 A6 can induce TFAP2β aggregation (phase separation) both in vivo and in vitro. (A) shows the virtual screening framework targeting TFAP2β molecules; (B) shows representative images of the FRAP experiment, displaying before bleaching (-3 s), during bleaching (0 s), and after bleaching (6 s), scale bar: 5 μm; (C) shows the quantitative analysis of the FRAP experimental results; (D, E) show the quantitative analysis of the number (D) and area (E) of TFAP2β spots in KYSE-150 cells after treatment with A6 or DMSO; (F) shows the droplet formation and precipitation results of His-GFP-TFAP2β under A6 or DMSO conditions, scale bar: 5 μm. μm; (G) is a detailed schematic of the interaction between compound A6 and TFAP2β (A chain); (H) is a comparison of the relative Gibbs free energy landscape between the TFAP2β-IDR2 bound to A6 state (TFAP2β-A6) and the ligandless state (TFAP2β-Apo), with the energy landscape axes representing the solvent-accessible surface area (SASA) and radius of gyration (Rg), respectively. Figure 13 A6 can induce TFAP2β aggregation (phase separation) both in vivo and in vitro. (A) HDX-MS Woods plot analysis of TFAP2β and R382A / N380A under A6 addition conditions; (B) SPR binding curves of unlabeled TFAP2β, R382A / N380A, and Mut_IDR2-FUS with A6; (CE) Droplet formation experiments (C) and quantitative analysis (D, E) of TFAP2β, R382A / N380A, or Mut_IDR2-FUS under A6 or DMSO addition conditions, scale bar: 5 μm; (F, G) dose-dependent effects of A6 on spot formation in cells expressing GFP-TFAP2β, GFP-R382A / N380A, or GFP-Mut_IDR2-FUS, scale bar: 5 μm. μm; (H, I) represent the effect of A6 / DMSO on the formation of TFAP2β and Mut_IDR2-FUS spots at pH=7 or 10: in vitro experiments (H) and in vivo experiments (I); Figure 14A6 inhibits ESCC progression by targeting and promoting TFAP2β aggregation; (AD) shows the dose-dependent effects of A6 on KYSE150 cell viability (A), apoptosis (B), migration (C), and invasion (D); (E) shows the correlation analysis between TFAP2β aggregate formation and cell viability in KYSE150, TE-1, and HET-1A cells at different A6 concentrations; (FI) shows the effects of A6 on proliferation (F), apoptosis (G), and migration / invasion (H and I) in WT and TFAP2β-KO KYSE150 cells; (J) is a schematic diagram of ESCC organoid establishment and drug treatment; (K) shows the H&E staining results of ESCC tissues and organoids, scale bar: 50 μm; (L) shows the effect of different concentrations of A6 on the cell viability of ESCC organoids; (M) shows the morphological images of organoids after treatment with A6 or DMSO, scale bar: 100 μm. μm; (N) shows the immunostaining results of TFAP2β (green) after adding A6 or DMSO to organoid cells, DAPI (blue), scale bar: 5 μm; (OQ) shows the quantitative analysis of TFAP2β fluorescence intensity (O), number of spots (P) and spot size (Q) of a single cell in Figure (N); Figure 15 This study demonstrates how A6 inhibits the progression of ESCC by targeting and promoting TFAP2β aggregation. (A, B) are schematic diagrams of the experimental procedure for A6 treatment in PDX mice (A) and subcutaneous xenograft mice (B); (C, D) are anatomical tumor images of PDX mice (C) and subcutaneous xenograft mice (D) after treatment with DMSO or A6; (E, F) are tumor growth curves of PDX mice (E) and subcutaneous xenograft mice (F) after treatment with DMSO or A6. Figure 16 A6 inhibits the progression of ESCC by targeting and promoting TFAP2β aggregation; (A) Immunostaining results of TFAP2β (green) in PDX tumors treated with DMSO or A6, DAPI (blue), scale bar: 5 μm; (BD) Quantitative analysis of TFAP2β fluorescence intensity (B), number of spots (C), and spot size (D) in single cells in Figure (A); (E) Immunostaining results of TFAP2β (green) in subcutaneous xenograft tumors treated with DMSO or A6, DAPI (blue), scale bar: 5 μm. μm; (FH) shows the quantitative analysis of TFAP2β fluorescence intensity (F), number of spots (G), and spot size (H) in Figure (E); (IJ) shows the effects of overexpression of GFP, GFP-TFAP2β, or GFP-R382A / N380A in WT or TFAP2β-KO KYSE150 cells, and treatment with A6 or DMSO, on cell viability (I), apoptosis (J), migration, and invasion (K, L); Figure 17 To discover new targets for phase separation in diseases and the drug mechanism of phase separation-targeted therapy. Detailed Implementation

[0013] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0014] Example 1 1. Perform combined ATAC-seq and RNA-seq analysis on tissue samples: Paired tissue samples (adjacent tissue P, mucosal carcinoma tissue Tm, and submucosal carcinoma tissue Tsm) were obtained from 28 patients with early-stage esophageal squamous cell carcinoma (ESCC) via endoscopic biopsy, and were analyzed using a combination of ATAC-seq and RNA-seq. Figure 1 A), to map chromatin accessibility and transcriptome overview in the early stages of ESCC.

[0015] The results showed that a total of 66 esophageal biopsy samples were obtained from 28 patients, including 28 P, 31 Tm, and 7 Tsm ( Figure 1 B), ATAC-seq maps were generated from 32 samples (14 P, 15 Tm, and 3 Tsm). Principal component analysis (PCA) revealed differences in chromatin accessibility between P and Tm, and between P and Tsm, but the differences between Tm and Tsm were small. Figure 2 A), a total of 17,250 differentially accessible peaks were identified between P and Tm (A). Figure 2 B). RNA-seq was performed on 34 samples (14 P, 16 Tm, and 4 Tsm). Compared with P, Tm showed 496 downregulated genes and 796 upregulated genes. Figure 2 C). Combined ATAC-seq and RNA-seq analysis identified 532 overlapping genes in Tm and P ( Figure 2 D), correlation analysis confirmed that changes in chromatin accessibility were positively correlated with differentially expressed genes in Tm and P (D), Figure 2 E). The crossover between Tm and P and Tsm and P explains one overlapping upregulated cerebellar TF zinc finger protein 2 (ZIC2) and three overlapping downregulated TFs (TFAP2β, NFIX, and ID4). Figure 2F). Considering existing reports of ZIC2, NFIX, and ID4 being associated with esophageal cancer, the focus was placed on TFAP2β, which showed reduced expression levels in Tm and Tsm tissues. Cancer Genome Atlas (TCGA) analysis revealed a significant negative correlation between TFAP2β expression and EC prognosis. Figure 2 G), indicating that TFAP2β may serve as a prognostic biomarker for EESCC (early ESCC). Figure 2 H). Therefore, combined ATAC-seq and RNA-seq analyses elucidated TFAP2β as a key downregulated TF in EESCC.

[0016] 2. TFAP2β increases apoptosis in ESCC while inhibiting cell proliferation, migration, and invasion. Immunohistochemistry (IHC), Western blot (WB), and reverse transcription quantitative PCR (RT-qPCR) were performed on paired specimens from 16 patients with esophageal squamous cell carcinoma. TFAP2β was overexpressed in HET-1A, TE-1, and KYSE150 cells, and changes in cell proliferation, invasion, migration, and apoptosis were detected. The effect of TFAP2β on ESCC was studied in two mouse models.

[0017] The results showed that the mRNA and protein levels of TFAP2β gradually decreased from the early to the late stages. Figure 3 A and Figure 3 B), the expression of TFAP2β in esophageal cancer cell lines TE-1 and KYSE150 was also lower than that in the normal human esophageal cancer cell line HET-1A (B). Figure 3 C). Overexpression of TFAP2β in TE-1 and KYSE150 cells showed that TFAP2β overexpression inhibited colony formation and cell proliferation. Figure 3 D-3G), TFAP2β knockout accelerates the proliferation of TE-1 and HET-1A cells ( Figure 3 H and Figure 3 I). Based on flow cytometry (FACS), scratch healing, and transwell assays, TFAP2β can also induce apoptosis and inhibit cell invasion and migration. Figure 3 J- Figure 3 L). The effects of TFAP2β on ESCC were investigated in two mouse models. Figure 4 A), in a mouse subcutaneous xenograft model, tumors formed by KYSE150 cells stably overexpressing GFP-TFAP2β were smaller than those in the GFP-overexpressing group (A). Figure 4 B and Figure 4 C). In the tail vein metastasis model, the control group showed more metastatic cell signaling than the TFAP2β overexpression group (C). Figure 4D), and also showed more metastatic nodules in the lungs and liver ( Figure 4 E- Figure 4 H). Therefore, TFAP2β levels gradually decrease during ESCC progression, which increases cell proliferation, migration, and invasion while inhibiting apoptosis.

[0018] 3. TFAP2β exhibits aggregation (phase separation) properties both in the cell nucleus and in vitro. Human TFAP2β and three other key ESCC TFs (ZIC2, NFIX, and ID4) were identified as containing intrinsically disordered regions (IDRs), which are key domains mediating liquid-liquid phase separation (LLPS). Immunostaining was performed on TFAP2β in P and Tm tissues, HET-1A, TE-1, and KYSE150 cells. Photobleaching recovery of fluorescence (FRAP) assay was performed in KYSE150 cells stably expressing GFP-TFAP2β. The presence of GFP-TFAP2β aggregates was detected under different pH conditions.

[0019] The results showed that TFAP2β formed droplet-like nuclear spots in P cells but not in Tm cells, which was positively correlated with its expression. Figure 5 A- Figure 5 D). Similarly, compared to KYSE150 or TE-1 cells, TFAP2β formed more nuclear spots and showed stronger immunofluorescence intensity in HET-1A cells (D). Figure 5 E- Figure 5 G). Then, in KYSE150 cells stably expressing GFP-TFAP2β, fluorescence recovery after photobleaching (FRAP) assays were performed, revealing that GFP-TFAP2β formed nuclear condensates similar to those of the endogenous protein, exhibiting high kinetics (G). Figure 5 H and Figure 5 I). The observed FRAP recovery could also be caused by dynamic binding to DNA, further demonstrating the aggregation behavior of TFAP2β. Experimental results show that GFP-TFAP2β aggregates diffuse between pH 7 and 10, and reform upon returning to pH 7, indicating that these nuclear spots are reversible. Figure 5 J). Correlation of light and transmission electron microscopy (CLEM) measurements revealed that cells transfected with GFP-TFAP2β exhibited membrane-free nuclear spots with high electron density. Figure 6 A). Based on dynamic light scattering (DLS) measurements, a sharp increase in the light scattering intensity of the GFP-TFAP2β protein was also observed. Figure 6B). Furthermore, purified human mCherry-TFAP2β, GFP-TFAP2β, and His-TFAP2β proteins co-formed droplets in vitro ( Figure 6 C and Figure 6 D); Both GFP-TFAP2β and mCherry-TFAP2β droplets exhibited fusion and kinetic behavior (D); Figure 6 E- Figure 6 J). Therefore, this indicates that TFAP2β exhibits aggregation (phase separation) properties both in the cell nucleus and in vitro.

[0020] 4. The aggregation (phase separation) properties of TFAP2β mediate its function in ESCC cell proliferation, apoptosis, and migration. Human TFAP2β has a proline / glutamine-rich (P / Q-rich) domain (29-119 aa), followed by a DNA-binding domain (DBD) and a helix-span-helix (HSH) motif (212-418 aa). Figure 7 A). Based on bioinformatics analysis and crystallographic studies of TFAP2β (219-433 aa), TFAP2β may contain two IDRs, including 34-220 aa (IDR1) and 433-460 aa (IDR2). Figure 7 A). Due to the overlap of TFAP2β-IDR1 with P / Q-rich and DBD ( Figure 7 A) The research focuses on IDR2. Different mutants of TFAP2β (ΔIDR1, ΔIDR2, Mut_IDR2, ΔIDR2-FUS, and Mut_IDR2-FUS, etc.) were constructed, and the aggregation behavior of different mutants was studied in vivo and in vitro.

[0021] ΔIDR2 exhibited reduced aggregation behavior in the nuclei of KYSE150 and TE-1 cells. Figure 7 C). Due to the influence of pH on TFAP2β aggregation and the fact that LLPS are typically regulated by electrostatic intermolecular interactions, sequence analysis of TFAP2β-IDR2 across different species revealed six conserved positively charged residues (lysine and arginine). By replacing these six residues with negatively charged glutamate, a TFAP2β mutant (Mut_IDR2) was constructed. Figure 7 A). Compared to WT, Mut_IDR2 showed reduced aggregation behavior both in vitro and in vivo. Figure 7 B and Figure 7C). Subsequently, ΔIDR2 or Mut_IDR2 was fused with FUS IDR (ΔIDR2-FUS and Mut_IDR2-FUS) to restore its aggregation behavior; both ΔIDR2-FUS and Mut_IDR2-FUS restructured the droplet aggregates. Figure 7 A- Figure 7 C). Then, wild-type TFAP2β (GFP-TFAP2β-WT), TFAP2β aggregation-deficient mutants (GFP-TFAP2β-ΔIDR2 or GFP-TFAP2β-Mut_IDR2), or TFAP2β aggregation-restoring mutants (GFP-TFAP2β-ΔIDR2-FUS or GFP-TFAP2β-Mut_IDR2-FUS) were overexpressed in KYSE150 or TE-1 cells. Compared with overexpression of GFP-TFAP2β-WT, overexpression of GFP-TFAP2β-ΔIDR2 or GFP-TFAP2β-Mut_IDR2 showed a reduced effect on antitumor activity, while GFP-TFAP2β-ΔIDR2-FUS or GFP-TFAP2β-Mut_IDR2-FUS restored tumor suppressor function. Figure 7 D- Figure 7 G). TFAP2β was knocked down in KYSE150 cells and overexpressed with TFAP2β nonsense mutants resistant to the targeting oligonucleotides (GFP-TFAP2β-NS, GFP-TFAP2β-ΔIDR2-NS, GFP-TFAP2β-Mut_IDR2-NS, GFP-TFAP2β-ΔIDR2-FUS-NS, or GFP-TFAP2β-Mut_IDR2-FUS-NS). Compared with overexpression of GFP-TFAP2β-NS, GFP-TFAP2β-ΔIDR2-NS, and GFP-TFAP2β-Mut_IDR2-NS, recovery was weakened in TFAP2β-knockdown cells for all three overexpression forms, while FUS-mediated aggregation recovery was restored. Figure 7 H- Figure 7 K).

[0022] Next, the effects of TFAP2β aggregation on mice were investigated. Figure 8 A). In a subcutaneous xenograft model, tumors formed by cells overexpressing green fluorescent protein (GFP), GFP-TFAP2β-ΔIDR2, or GFP-TFAP2β-Mut_IDR2 were larger than those formed by cells overexpressing GFP-TFAP2β. Tumors formed by cells overexpressing GFP-TFAP2β-ΔIDR2-FUS or GFP-TFAP2β-Mut_IDR2-FUS were smaller than those formed by cells overexpressing the corresponding mutants lacking aggregation function. Figure 8 B and Figure 8 C). GFP-TFAP2β and two GFP-TFAP2β aggregation recovery variants (GFP-TFAP2β-ΔIDR2-FUS or GFP-TFAP2β-Mut_IDR2-FUS) formed typical droplet-like dots in the nuclei of tumor cells. Figure 8 D). In the tail vein metastasis model, TFAP2β agglutination reduced tumor metastasis (D). Figure 8 E- Figure 8 I). Therefore, this indicates that TFAP2β aggregation is crucial for inhibiting tumor growth and metastasis in vivo.

[0023] 5. TFAP2β condensates inhibit ZNF131 transcription to suppress ESCC and recruit other key TFs to promote their DNA binding. Previous studies have shown that TF aggregates can mediate TF transcriptional function, so this study aimed to determine whether and how TFAP2β aggregates affect the DNA binding capacity of TFAP2β and its downstream regulatory functions. To identify the direct downstream targets of TFAP2β aggregates, ChIP-seq and RNA-seq data were analyzed; fluorescence in situ hybridization (FISH) and single-molecule fluorescence in situ hybridization (smFISH) experiments were performed to observe the intracellular behavior of TFAP2β; and ChIP-qPCR was used to analyze the interaction between TFAP2β and the ZNF131 promoter, as well as the regulatory relationship between TFAP2β and ZNF131.

[0024] Experimental results showed that comparing GFP-TFAP2β with GFP-TFAP2β-Mut_IDR2 or GFP-TFAP2β-ΔIDR2 in ChIP-seq and RNA-seq data, only one gene, ZNF131, was found in these five datasets. Figure 9 A). Subsequently, by visualizing ZNF131, and using a gene browser containing ChIP-seq and RNA-seq data, it was confirmed that the TFAP2β condensate binds to its promoter region (chromosome 5: 43,066,767-43,067,712). Figure 9 B). To further confirm this, fluorescence in situ hybridization (FISH) and single-molecule fluorescence in situ hybridization (smFISH) experiments were performed, and co-localization of the GFP-TFAP2β body with the ZNF131 promoter focus in the nucleus was observed. Figure 9C). ChIP-qPCR analysis further showed that GFP-TFAP2β interacts with the ZNF131 promoter, while the binding affinity of GFP-TFAP2β-ΔIDR2 and GFP-TFAP2β-Mut_IDR2 is reduced, and this reduction is reversed by their corresponding FUS fusion proteins. Figure 9 D). Similar phenomena were also observed at the ZNF131 mRNA level and transcriptional levels using RT-qPCR and luciferase assays. Figure 9 E and Figure 9 F).

[0025] Since ZNF131 has been identified as an oncogene, this study investigates whether it acts as a downstream function of TFAP2β in regulating the pathogenesis of esophageal squamous cell carcinoma (ESCC). ZNF131 overexpression alleviates the antitumor effect of TFAP2β overexpression. Figure 10 A- Figure 10 D), while ZNF131 RNAi reversed the knockdown effect of TFAP2β during ESCC development ( Figure 10 E- Figure 10 H). Although TFAP2β-IDR1 contains a P / Q-rich domain, which is generally associated with transcriptional activation, transcription factors (TADs) containing the transcriptional activation domain occasionally repress transcription. In hepatocellular carcinoma, Yes-associated protein 1 (YAP1) has been identified as a transcriptional activator that binds directly to the ZNF131 promoter. Experimental results showed that YAP1 overexpression also increased the mRNA level and transcriptional activity of ZNF131 in KYSE150 cells. Figure 10 I and Figure 10 J). Furthermore, ChIP-qPCR analysis revealed that TFAP2β and its aggregation behavior inhibited the binding of YAP1 to the ZNF131 promoter region in ESCC cells (J). Figure 10 Therefore, the TFAP2β condensate reduces ZNF131 transcription by inhibiting the binding of YAP1 to the ZNF131 promoter region.

[0026] ESCC cell lysates were added to droplets formed by GFP-TFAP2β or GFP-FUS, and droplet centrifugation experiments were performed. It was found that two other key downregulated transcription factors (NFIX and ID4) were enriched in GFP-TFAP2β, but not in the GFP-FUS droplet precipitate. Figure 11 A and Figure 11 B). Co-localization behavior between GFP-TFAP2β and mCherry-NFIX punctate groups was observed by in vivo imaging. Figure 11 C and Figure 11D). It is previously known that NFIX binds to the Meis1 enhancer region in ESCC cells, and overexpression of GFP-TFAP2β or GFP-TFAP2β-Mut_IDR2-FUS results in a stronger binding affinity between NFIX and the Meis1 enhancer than that of GFP-TFAP2β-Mut_IDR2. Figure 11 E). Therefore, the TFAP2β condensate serves as a platform for recruiting other key transcription factors (NFIX and ID4) to promote their transcriptional activity in ESCC.

[0027] 6. A6 can induce the aggregation (phase separation) of TFAP2β both in vivo and in vitro. Based on a complex compound library of various transcription factor-targeting drugs, a customized virtual screening framework was constructed, identifying 16 small molecules ( Figure 12 A). Among them, compound A6 increased the dynamic formation and aggregation of GFP-TFAP2β aggregates both in vivo and in vitro. Figure 12 B- Figure 12 F). Molecular dynamics simulations show that the binding groove of TFAP2β is located in the HSH helical region, which is close to the DBD groove (377-383 aa). Notably, Arg382 forms a stable π-cation interaction with the benzimidazole ring, while Asn380 forms a π-lone pair interaction with the aromatic ring on the opposite side of A6. Figure 12 G). Since TFAP2β-IDR2 is mainly associated with the co-condensation of TFAP2β and functions in ESCC, its free energy spectrum was further plotted using soluble surface area (SASA) and radius of gyration (Rg). The binding of A6 induced a more stable and ordered folding conformation of IDR2 on chains A and B. Figure 12 H).

[0028] Next, hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis was performed, and it was observed that the exchange rate of TFAP2β (219-460 aa) in the presence of A6 was significantly reduced in four regions compared to the inactive state: residues 242-251, 270-309, 372-397, and 427-460 (IDR2). Figure 13 A). Since Arg382 and Asn380, as determined in molecular simulations, are located within residues 372-397, they are likely two key binding sites. Therefore, these two key residues were mutated to alanine (R382AN380A) to assess whether A6 promotes the liquid self-assembly of TFAP2β through direct interaction. The binding affinity of wild-type TFAP2β to A6 was much stronger than that of R382AN380A, which was confirmed by surface plasmon resonance (SPR) testing. Figure 13 B). Furthermore, ΔIDR1 or ΔIDR2 did not significantly affect the binding affinity of A6 compared to the full-length TFAP2β. Figure 13 B). This indicates that A6 binds directly to the DBD-HSH region via Arg382 and Asn380, rather than through the disordered region. The conformational change at 427-460 aa (IDR2) is indirectly caused by A6 binding based on HDX-MS data. To verify this, HDX-MS measurements were performed on R382AN380A (219-460 aa). Based on this, R382AN380A exhibits the same exchange pattern under A6 addition conditions, i.e., the decrease in exchange rate in regions 242-251, 270-309, 372-397, and 427-460 (IDR2) disappears together. Figure 13 A). Droplet formation of WT TFAP2β, Mut_IDR2-FUS, and R382AN380A was observed by adding A6; correspondingly, in vivo and in vitro, compared with WT TFAP2β, the addition of A6 did not induce aggregation of either Mut_IDR2-FUS or R382AN380A. Figure 13 C- Figure 13 G). SPR data showed that the binding affinity of Mut_IDR2-FUS to A6 was similar to that of WT TFAP2β (G). Figure 13 B). Furthermore, at pH 7, A6 promoted the coagulation of WT TFAP2β, but did not have this effect on Mut_IDR2-FUS. At pH 10, the addition of A6 did not enhance the coagulation of either TFAP2β or Mut_IDR2-FUS. Figure 13 H and Figure 13 I). Therefore, these results all indicate that A6 directly interacts with TFAP2β through Arg382 and Asn380, promoting its aggregation through electrostatic intermolecular interactions and conserved positively charged residues in IDR2.

[0029] 7. A6 inhibits the progression of ESCC by targeting and inducing TFAP2β aggregation. The study validated that A6 can inhibit the proliferation, migration, and invasion of esophageal squamous cell carcinoma (ESCC) cells in a dose-dependent manner and promote apoptosis. Figure 14 A- Figure 14 D). Furthermore, consistent with the differences in TFAP2β expression, normal esophageal epithelial cells required higher A6 concentrations compared to ESCC cells to reduce cell viability and enhance TFAP2β aggregation (D). Figure 14E). TFAP2β gene knockout (KO) significantly attenuated the tumor-suppressive effect of A6, indicating that TFAP2β is a target of A6 in its anti-tumor function. Figure 14 F- Figure 14 I).

[0030] Organoids provide a platform for evaluating drug efficacy and toxicity, and drug responses observed in human ESCC-derived organoids are highly consistent with clinical responses in patients. Patient-derived ESCC organoids were established to further confirm the therapeutic effect of A6. Figure 14 J and Figure 4 K). Accordingly, the addition of A6 reduced the viability and size of organoid cells (K). Figure 14 L and Figure 14 Furthermore, A6 enhanced TFAP2β nucleus site formation but did not affect its expression (M). Figure 14 N- Figure 14 Q). By using patient-derived tumor xenograft (PDX) ( Figure 15 A) and subcutaneous xenograft ( Figure 15 B) Mouse models further confirmed the anti-tumor effect of A6 in vivo. A6 treatment significantly inhibited tumor growth in both mouse models without affecting body weight or causing significant organ toxicity to the heart, liver, lungs, and kidneys. Figure 15 C- Figure 15 F). Furthermore, the addition of A6 to both types of tumor cells increased nuclear aggregation of TFAP2β without affecting its protein levels. Figure 16 A- Figure 16 H).

[0031] Overexpression of GFP, GFP-TFAP2β, or GFP-R382AN380A in TFAP2β-KO cells revealed that TFAP2β effectively restored the ability of A6 to inhibit proliferation, migration, and invasion, as well as promote apoptosis, while R382AN380A exhibited a weakening effect. Figure 16 I- Figure 16 (L), indicating that A6 exerts its antitumor effect by directly targeting TFAP2β. In summary, the results suggest that TFAP2β forms nuclear condensates, serving as a platform to recruit other key transcription factors and promotes its own and its bound transcription factors' transcriptional activity, thereby regulating cell proliferation, apoptosis, migration, and invasion in esophageal squamous cell carcinoma (ESCC).

[0032] In summary, based on the previously established ATAC-seq library preparation method, this invention improved it and applied it to the study of chromatin accessibility in esophageal squamous cell carcinoma tissues. Through comprehensive analysis combining ChIP-seq, ATAC-seq, and RNA-seq, it was demonstrated that TFAP2β aggregation regulates tumorigenesis and transcription in ESCC through its dual function. Figure 17 They also discovered a compound A6 induced by LLPS that exerts anti-tumor functions in cell, mouse, and patient-derived organoids, suggesting a potential clinical treatment strategy for LLPS-mediated ESCC.

[0033] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. The use of compound A6 in the preparation of drugs for treating esophageal squamous cell carcinoma, wherein the structural formula of compound A6 is shown in Formula I. 。 2. The application according to claim 1, characterized in that: The drug comprises compound A6 and / or its pharmaceutically acceptable salt.

3. The application according to claim 2, characterized in that: The drug is in the form of tablets, capsules, powders, pills, granules, gels, injections, or emulsions.

4. The application according to claim 1, characterized in that: The esophageal squamous cell carcinoma was caused by KYSE150 cells or TE-1 cells.

5. The application according to claim 1, characterized in that: The anti-esophageal squamous cell carcinoma method includes promoting apoptosis of esophageal squamous cell carcinoma cells and inhibiting the proliferation, invasion, and / or migration of esophageal squamous cell carcinoma cells.

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