Use of sulfasalazine in combination with a lactate dehydrogenase inhibitor for the preparation of a TIF1 gamma lactate product

Abstract

The present application relates to the application of sulfasalazine combined with lactate dehydrogenase inhibitor in preparing the inhibition of TIF1 gamma lactic acid product, belongs to the field of biological medicine technology. The present application finds that sulfasalazine can block the lactic acidification of the 763th and 769th lysine of transcription intermediate factor 1 gamma (TIF1 gamma), and the combination of sulfasalazine and lactate dehydrogenase inhibitor can strengthen the block of the lactic acidification of the 763th and 769th lysine of transcription intermediate factor 1 gamma, improve the expression level of TIF1 gamma, and then improve the treatment effect on TIF1 gamma related cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of sulfasalazine in combination with lactate dehydrogenase inhibitors in the preparation of products that inhibit TIF1γ lactation. Background Technology

[0002] In recent years, the interaction between tumor metabolic reprogramming (such as the Warburg effect) and protein post-translational modification (PTM) has become a research hotspot. Lactic acid, a metabolic product of glycolysis, is not only a byproduct of energy metabolism, but can also mediate novel protein lactation modifications. By altering the function of key proteins, it forms a "metabolism-modification-function" regulatory axis, thereby affecting the malignant progression of tumors.

[0003] TIF1γ (Transcriptional Intermediary Factor 1 gamma, also known as TRIM33) is a transcriptional regulator with E3 ubiquitin ligase activity, primarily playing a tumor-suppressive role in cancer treatment. It inhibits epithelial-mesenchymal transition (EMT) and the maintenance of tumor stem cell characteristics by negatively regulating the transforming growth factor-β (TGF-β) / Smad signaling pathway, thereby preventing tumor invasion and metastasis. In various solid tumors such as liver cancer, colorectal cancer, and pancreatic cancer, downregulation of TIF1γ expression is closely associated with poor patient prognosis, making it a potential prognostic biomarker. Furthermore, restoring TIF1γ expression or enhancing its function could be a novel strategy for cancer treatment, such as targeting the TGF-β pathway in combination with immunotherapy, or utilizing its ability to regulate the tumor microenvironment to improve the sensitivity of existing therapies. However, TIF1γ is often expressed at low levels in cancer and is easily degraded; simply increasing its expression is easily counteracted by high levels of lactate in the tumor microenvironment, leading to poor therapeutic effects from targeted TIF1γ expression enhancement strategies. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem that the high expression effect of TIF1γ in the prior art is easily offset by the high concentration of lactic acid in the tumor microenvironment.

[0005] To address the aforementioned technical problems, this invention provides the application of sulfasalazine in combination with a lactate dehydrogenase inhibitor in the preparation of products that inhibit TIF1γ lactation. This invention discovers a pathological loop of "lactate accumulation-TIF1γ lactation-protein degradation-tumorigenesis," and significantly reverses the malignant positive feedback between tumor metabolism and epigenetic modification through a dual intervention strategy: inhibiting lactate production with a lactate dehydrogenase inhibitor (LDHi) (substrate-terminal shaving) and competitively binding to TIF1γ lactation modification sites with sulfasalazine (SAS) (protein-terminal blocking). Specifically, this invention has discovered that sulfasalazine can block the lactation of lysine residues at positions 763 and 769 of transcription mediator 1γ (TIF1γ). Combining sulfasalazine with a lactate dehydrogenase inhibitor can enhance the blocking of lactation of lysine residues at positions 763 and 769 of transcription mediator 1γ (TIF1γ), increase the expression level of TIF1γ, and thus improve the therapeutic effect on TIF1γ-related cancers.

[0006] The first objective of this invention is to provide an application of sulfasalazine in combination with a lactate dehydrogenase inhibitor in the preparation of a transcriptional mediator 1γ lactation blocking product, wherein the transcriptional mediator 1γ lactation blocking product targets and blocks the lactation of lysine residues at positions 763 and 769 of transcriptional mediator 1γ.

[0007] Furthermore, the lactate dehydrogenase inhibitor includes GSK2837808A.

[0008] Furthermore, the NCBI number of the transcriptional mediator 1γ is NP_056990.3.

[0009] A second objective of the present invention is to provide a pharmaceutical composition that blocks the lactation of lysine residues at positions 763 and 769 of transcription mediator 1γ, said pharmaceutical composition comprising sulfasalazine and a lactate dehydrogenase inhibitor.

[0010] A third objective of this invention is to provide the use of sulfasalazine in the preparation of a product that enhances the expression of transcription mediator 1γ, said product enhancing the expression of transcription mediator 1γ by blocking lactation of lysine residues at positions 763 and 769 of transcription mediator 1γ.

[0011] A fourth objective of this invention is to provide the use of a substance that inhibits lactation at positions 763 and 769 of transcription mediator 1γ in the preparation of a cancer treatment product, said substance targeting and inhibiting lactation of lysine residues at positions 763 and 769 of transcription mediator 1γ.

[0012] Furthermore, the substance includes sulfasalazine and / or lactate dehydrogenase inhibitors.

[0013] Furthermore, the cancers mentioned include lung cancer.

[0014] Furthermore, the lung cancer includes lung adenocarcinoma.

[0015] Furthermore, when the substance comprises sulfasalazine and lactate dehydrogenase inhibitor, the dosage ratio of sulfasalazine to lactate dehydrogenase inhibitor is 60-70 mg / kg: 2-4 mg / kg.

[0016] Furthermore, when the substance comprises sulfasalazine and a lactate dehydrogenase inhibitor, the dosage of sulfasalazine is 20-30% of the safe dosage of sulfasalazine alone, and the dosage of the lactate dehydrogenase inhibitor is 45-55% of the safe dosage of lactate dehydrogenase inhibitor alone.

[0017] A fifth objective of this invention is to provide the use of sulfasalazine and lactate dehydrogenase inhibitors in the preparation of a product that inhibits cancer metastasis, said product inhibiting the metastasis of cancer cells.

[0018] Furthermore, the product inhibits the metastasis of lung adenocarcinoma cells to the liver and spleen.

[0019] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0020] This invention utilizes mass spectrometry identification and site-directed mutagenesis to precisely pinpoint two core modification sites, K763 and K769, clarifying that sulfasalazine (SAS) specifically binds to the lactation-sensitive pocket of the TIF1γ protein composed of residues such as G50, R52, and E762. This precise binding mode targeting specific protein post-translational modification sites creates steric hindrance, competitively blocking the addition of lactyl groups, providing clear structural biological evidence for precision clinical treatment. Furthermore, combining sulfasalazine with a lactate dehydrogenase inhibitor enhances this blocking effect. In a spontaneous lung cancer model of KSLE, the combination therapy reduced the number of lung tumor nodules by more than 80% and successfully blocked the formation of micrometastases from cancer cells to distant organs such as the liver and spleen. Attached Figure Description

[0021] Figure 1The first figure shows the results of bioinformatics analysis of low TIF1γ expression in LUAD and its correlation with prognosis, highlighting its close association with glycolytic metabolism. Figure A shows the expression of TIF1γ in LUAD and LUSC tumors and normal tissues using the TCGA and GTEx databases. Figure B shows the relationship between TIF1γ expression in LUAD and LUSC and patient prognosis. Figure C shows the results of quality control, dimensionality reduction, and cluster analysis of the lung adenocarcinoma single-cell dataset GSE189357, evaluating the expression of TIF1γ in the early non-invasive and invasive groups of lung adenocarcinoma. TD-1 to TD-9 represent cases 1-9, where n represents the sample size, and *** indicates... P < 0.001, the same applies below;

[0022] Figure 2 The first figure shows the results of low TIF1γ expression in LUAD cells and its function being closely related to glycolytic metabolism; AB shows the protein expression levels of TIF1γ in normal human bronchial epithelial cell lines, LUAD cell lines, A549 untransferred cells, A549 lung metastases, and A549 bone metastases, with β-actin as an internal control; C shows the results of mass spectrometry (MS) and transcriptome sequencing analysis; DE shows the results of GO / KEGG enrichment analysis of the impact of TIF1γ on the biological processes of LUAD, BP represents biological processes, CC represents cellular components, and MF represents molecular functions.

[0023] Figure 3 The first graph shows the results of TIF1γ low expression driving glycolytic metabolic reprogramming and promoting lactate production; AE shows the glycolytic rate curves, basal glycolytic rate, and compensatory glycolytic rate of A549 and H1650 cells after TIF1γ knockdown; FG shows the heatmap and volcano plot of differential metabolites detected by targeted energy metabolomics in A549 cells after TIF1γ knockdown; H shows the lactate content in the intracellular and culture supernatant of A549 and H1650 cells in the control and TIF1γ knockdown groups; where ** indicates P < 0.01, the same applies below;

[0024] Figure 4The diagram shows the results of lactate-induced TIF1γ lactation modification and its effects on protein stability and spatial distribution; A is a schematic diagram of TIF1γ lactation modification; B is a diagram showing the results of TIF1γ lactation modification confirmed by anti-TIF1γ immunoprecipitation and pan-lactation antibody detection in HEK293T cells overexpressing TIF1γ; C is a diagram showing the results of TIF1γ lactation modification further verified by endogenous immunoprecipitation in A549 cells; D shows that treatment with lactate (10 mM) and sodium lactate (10 mM) enhances TIF1γ lactation modification, while treatment with lactate dehydrogenase inhibitor LDHi (10 μM) and glycolysis inhibitor 2-DG (8 mM) inhibits this modification; EF shows the results of treating A549 and H1650 cells with different gradient concentrations of lactate or LDHi for 24 hours, and the gradient time with a fixed concentration (lactic acid: 10 mM; LDHi: 10 mM). The results of TIF1γ protein expression level detection by Western blotting after cell treatment with HA-TIF1γ (10 mM) were shown, with β-actin as an internal control; GH showed the results of TIF1γ lactation modification level detection by anti-HA immunoprecipitation and pan-lactic acidification modification antibody detection after HEK293T cells were overexpressed with HA-TIF1γ and treated with different gradient concentrations of lactate or LDHi for 24 hours, and after different gradient times and fixed concentrations (lactic acid: 10 mM; LDHi: 10 μM); I showed the results of A549 and H1650 cells treated with or without lactate (10 mM, 24 h), incubated with rabbit anti-TIF1γ antibody, stained with fluorescent dye Cy3-conjugated anti-rabbit IgG (immunoglobulin G), and counterstained with 4',6-diamidinyl-2-phenylindole (DAPI, blue); J showed the results of nucleocytoplasmic separation experiment verifying lactate (10 mM, 24 h)... h) The results of treating A549 and H1650 cells to transfer the subcellular localization of TIF1γ from the nucleus to the cytoplasm. Lamin B1 is used as a positive control for nuclear proteins, and β-actin is used as a positive control for cytoplasmic proteins. "+" indicates addition, and "-" indicates no addition. The same applies below.

[0025] Figure 5A) Identification and dynamic regulation verification of lactation modification at the K763 / K769 sites of TIF1γ; B) Mass spectrometry identification of lactation modification of TIF1γ peptide at the K763 / K769 sites; C) Verification results of lactation modification levels after transfection of wild-type TIF1γ and TIF1γ mutant in HEK293T cells; D) Species conservation analysis of TIF1γ-K763 and TIF1γ-K769 sites; E) Verification of the specific recognition ability of Kla763-modified antibody and Kla769-modified antibody for lactated peptide; F) Results of endogenous immunoprecipitation in A549 and H1650 cells and detection of lactation modification of TIF1γ using Kla763- and Kla769-modified antibodies; G1) Detection of lactation modification of wild-type and 2KR mutant TIF1γ at lactate (10 mM) and LD1 (10 mM) using immunoprecipitation experiments. Figure 1 shows the detection results of lactation modification levels at K763 / K769 sites after 24 hours of TIP60 overexpression treatment with μM or TIP60;

[0026] Figure 6 The first image shows the results of a virtual screening of sulfasalazine targeting TIF1γ to inhibit its lactation modification; A is a superimposed three-dimensional structure of wild-type TIF1γ-WT constructed using AlphaFold3 and simulated lactation-modified TIF1γ-Kla763 / 769 (hereinafter referred to as TIF1γ-Kla); B is a graph showing the results of root mean square deviation (RMSD) and root mean square fluctuation (RMSF) analysis of TIF1γ-WT and TIF1γ-Kla763 / 769 under 1000 ns all-atom molecular dynamics simulation, evaluating the impact of lactation modification on protein conformational stability; C is a flowchart of a virtual screening of the DrugBank drug library targeting the pocket near the K763 / K769 site; D is a 1000 ns all-atom molecular dynamics simulation. RMSD and RMSF analyses of TIF1γ-WT-unloaded state (ligand-free state) and TIF1γ-WT-loaded state (SAS-bound state) under ns all-atom molecular dynamics simulations; E is a graph showing the binding mode of SAS and TIF1γ and the key hydrogen bond interaction amino acid residues through molecular docking, with the binding free energy calculated by MM / GBSA annotated.

[0027] Figure 7The first graph shows the results of sulfasalazine and LDHi synergistically blocking cancer progression; A and B are the kinetic curves and dissociation constants of SAS binding to TIF1γ protein detected by surface plasmon resonance (SPR) assay; C is the results of immunoprecipitation (IP) detection of the effects of SAS and LDHi on the lactation level of TIF1γ protein K763 / K769 in HEK293T cells; D is the results of Western blot detection of changes in TIF1γ protein level and EMT marker expression under different treatment groups, with β-actin as an internal control; E is the detection of the effect of SAS on EMT marker expression in control group, TIF1γ knockdown or TIF1γ replenishment cancer cells, to assess the drug dependence of SAS, with β-actin as an internal control.

[0028] Figure 8 The first graph shows the synergistic inhibitory effect of different concentrations of SAS and LDHi combined on the in vitro metastasis of cancer cells; AD represents the Transwell migration and invasion assay, which detected the effects of different concentrations of SAS and LDHi alone (SAS: 0-200 μM; LDHi: 0-10 μM) or in combination on the migration and invasion abilities of A549 and H1650 cells; EF represents the scratch healing assay, which detected the migration rate of A549 and H1650 cells within 24 hours under the above administration conditions. * indicates... P < 0.05, ** indicates P <0.01, *** indicates P < 0.001;

[0029] Figure 9 The results are shown in Figure 1. A is the result of the combination of sulfasalazine and LDHi significantly reducing tumor micrometastases in a mouse lung cancer model. Figure 2 is the flowchart of the experimental design and dosing regimen. Figure 3 is the result of tumor burden in the lungs of mice in the control group and the combined treatment group after one month of treatment, detected by a small animal in vivo imaging system. Figure 4 is the result of Bouin's staining and the statistical graph of the number of tumor nodules. Figure 5 is the in vivo imaging and statistical results of tumor metastasis. Figure 6 is the H&E staining and statistical graph of the number of tumor nodules in the lungs, liver and spleen. Figure 7 is the statistical results of the effect of drug combination on the body weight of mice. Detailed Implementation

[0030] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0031] The spontaneous lung cancer mouse model (KSLE mouse model) involved in the following examples was purchased from Shanghai Southern Model Biotechnology Co., Ltd., catalog number nm-cko-234719.

[0032] Example 1: Detection of Tumor Suppressor Targets and Elucidation of Metabolic Reprogramming Mechanisms

[0033] This embodiment aims to establish the biological role of transcriptional mediator 1γ (TIF1γ) in lung adenocarcinoma metastasis and to analyze the abnormal glycolytic metabolism caused by its deficiency, so as to provide a theoretical basis for subsequent precise intervention.

[0034] First, transcriptome sequencing data from lung adenocarcinoma (n=892) and normal tissues were extracted and analyzed from the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) databases. The results showed that TIF1γ mRNA was significantly downregulated in lung adenocarcinoma (LUAD) tissues compared with normal tissues. Figure 1 (A). Subsequently, the relationship between TIF1γ expression abundance and patient prognosis was assessed using the survival curve plotting tool (Kaplan-Meier Plotter). Clinical survival statistics confirmed that the overall survival time of the high TIF1γ expression group was significantly longer than that of the low expression group, while this phenomenon was not significant in lung squamous cell carcinoma (LUSC), thus defining the specific anti-tumor value of TIF1γ in lung adenocarcinoma. Figure 1 B).

[0035] To investigate the relationship between TIF1γ and tumor malignant progression at the single-cell level, quality control, dimensionality reduction, and cluster analysis were performed on the lung adenocarcinoma single-cell dataset GSE189357. Tumor epithelial cell subsets were extracted and compared, revealing that the expression level of TIF1γ was significantly pathologically reduced after lung adenocarcinoma entered the invasive stage (IAC) compared to the early non-invasive stage (AIS+MIA). Figure 1 C). Further confirmation at the cellular level was achieved by using Western blot (WB) to detect human normal bronchial epithelial cells (HBE), human bronchial epithelial cells (BEAS-2B), human lung adenocarcinoma cells (A549), and human non-small cell lung cancer cells (H1650). The results showed that TIF1γ expression in LUAD cells was significantly lower than in normal cells, and its expression was further lost in the A549 subline with lung or bone metastases, confirming a negative correlation between its protein abundance and metastatic ability. Figure 2 (AB).

[0036] After clarifying the tumor-suppressive characteristics of TIF1γ, we explored its regulatory mechanism using multi-omics interaction analysis. We identified 104 potential interacting proteins by immunoprecipitation combined with mass spectrometry (MS) on A549 cells overexpressing Flag-TIF1γ (TIF1γ with a Flag tag). These interacting proteins were then combined with 4693 differentially expressed genes screened by transcriptome sequencing. Figure 2According to the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, TIF1γ was significantly enriched in glucose metabolism and pyruvate metabolism. Figure 2 (DE).

[0037] Subsequently, the effect of TIF1γ on glycolytic metabolism was measured using a Seahorse XF energy metabolism analyzer. The experiment was first divided into six groups and treated as follows:

[0038] A549 si-NC group: A549 cells without TIF1γ knockout;

[0039] A549 si-TIF1γ-1 group: si-TIF1γ-1 (si-TIF1γ-1 was prepared by introducing a TT pendant structure at the end of the sequence shown in SEQ ID NO.1 (CGACAUCCAGCAAGUUGGU)) was introduced into A549 cells to knock out TIF1γ;

[0040] A549 si-TIF1γ-2 group: si-TIF1γ-2 (prepared by introducing a TT pendant structure into the end of the sequence shown in SEQ ID NO.2 (AGUCAGGGCUCAGCAGCCU)) was introduced into A549 cells to knock out TIF1γ;

[0041] H1650 si-NC group: H1650 cells did not have TIF1γ knocked out;

[0042] H1650 si-TIF1γ-1 group: si-TIF1γ-1 (si-TIF1γ-1 was prepared by introducing a TT pendant structure at the end of the sequence shown in SEQ ID NO.1 (CGACAUCCAGCAAGUUGGU)) was introduced into H1650 cells to knock out TIF1γ;

[0043] H1650 si-TIF1γ-2 group: si-TIF1γ-2 (prepared by introducing a TT pendant structure at the end of the sequence shown in SEQ ID NO.2 (AGUCAGGGCUCAGCAGCCU)) was introduced into H1650 cells to knock out TIF1γ.

[0044] Based on the knockdown of TIF1γ in A549 and H1650 cells, this embodiment selected the si-TIF1γ-2 group and the si-NC group, which showed better knockdown effects, for subsequent experiments. Figure 3The four groups of cells were cultured, and at approximately 20 minutes, rotenone / antimycin A (Rot / AA, 0.5 μM) was injected into the cells. At approximately 45 minutes, 2-deoxy-D-glucose (2-DG, a glycolysis inhibitor, 50 mM) was injected. The glycolysis rate and compensatory glycolysis rate were monitored in real time using a Seahorse XFe96 energy metabolism analyzer. The experimental results showed that both the basal glycolysis rate and the compensatory glycolysis rate of the cells were significantly increased. Figure 3 (CE).

[0045] To confirm the changes in metabolites, targeted energy metabolomics was performed on the aforementioned cells for validation. The differential metabolite heatmap and volcano plot visually demonstrated that TIF1γ deficiency led to a significant accumulation of pyruvate and lactate levels in LUAD cells. Figure 3 Finally, the L-lactic acid was precisely quantified using the L-lactic acid colorimetric method, and the results confirmed that after TIF1γ knockdown, the lactate content in both intracellular fluid and culture supernatant increased exponentially. Figure 3 H).

[0046] Example 2: Identification of TIF1γ core modification sites and mechanism blocking sites

[0047] This embodiment utilizes post-translational modification detection technology and site-directed mutagenesis strategy to accurately locate the physical site that lactate regulates the stability of TIF1γ protein and elucidates the molecular pathological mechanism by which it induces loss of protein function.

[0048] First, TIF1γ was overexpressed in human embryonic kidney cells (HEK293T cells, hereinafter referred to as 293T cells) using endogenous and exogenous immunoprecipitation (IP) experiments (an expression vector pCDNA3.1-TIF1γ-HA containing TIF1γ and HA tags was constructed, with pCDNA3.1-HA vector as a blank control). The expression was then detected using a pan-lactation modification antibody (pan-Kla, brand name: PTMBIO, catalog number: PTM-1401RM, dilution ratio 1:1000). The results showed that TIF1γ protein exhibited a clear lactation modification band ( Figure 4 (B), and the presence of this modification was also confirmed in the endogenous system of A549 cells. Figure 4 (C, immunoglobulin G (lgG) was a negative control).

[0049] To verify the driving role of the metabolic environment in modification, 293T cells were divided into six groups and treated with dimethyl sulfoxide (DMSO), lactate, sodium lactate, the lactate dehydrogenase inhibitor LDHi, and the glycolysis inhibitor 2-DG, respectively. 293T cells without DMSO served as the control group. The modification level of TIF1γ was detected using a pan-lactation modification antibody. The results showed that increased lactate levels significantly enhanced pan-lactation modification of TIF1γ, while inhibition of lactate production significantly downregulated this modification level. Figure 4 D).

[0050] A549, H1650, and 293T cells were treated with different concentration gradients of lactate and the lactate dehydrogenase inhibitor LDHi, respectively, to observe the effects of different concentration gradients of lactate and LDHi on TIF1γ protein levels and lactation. Simultaneously, A549, H1650, and 293T cells were treated with 10 mM lactate or 10 μM LDHi for 0, 4, 8, 12, and 24 hours, respectively, to observe the effects of different treatment durations on TIF1γ protein levels and lactation.

[0051] Gradient concentration and time-series experiments further confirmed that TIF1γ protein levels decreased in a gradient manner with increasing lactate-induced lactation. Figure 4 (EH).

[0052] A549 and H1650 cells were divided into two groups and treated with lactate and dimethyl sulfoxide, respectively. Immunofluorescence and nucleoplasmic separation experiments revealed that in the dimethyl sulfoxide group, TIF1γ was mainly located in the nucleus, performing tumor-suppressive functions, while lactate stimulation significantly induced the translocation of TIF1γ from the nucleus to the cytoplasm. Figure 4 This leads to a vicious cycle of "lactic acid accumulation - modification enhancement - nuclear export - protein degradation".

[0053] To pinpoint the precise target of intervention, high-resolution liquid chromatography-tandem mass spectrometry (LC-MS / MS) was used to perform deep sequencing of the TIF1γ peptide, precisely identifying two core lactation-modified residues within its M domain: lysine at position 763 (K763) and lysine at position 769 (K769). Figure 5 Based on this finding, a site-specific K→R (i.e., lysine mutated to arginine) mutant plasmid was constructed using point mutation technology to simulate its delactation state.

[0054] Recombinant vectors containing Flag-TIF1γ-K763R, Flag-TIF1γ-K769R, and the dual-site mutant Flag-TIF1γ-2KR (both K763 and K769 are mutated to arginine) were constructed, with a blank vector and a recombinant vector containing Flag-TIF1γ as controls. Transfection into 293T cells and immunoprecipitation assays confirmed that, compared with wild-type (i.e., 293T cells containing the recombinant vector containing Flag-TIF1γ), the lactation signal of the dual-site mutant 2KR (i.e., TIF1γ containing both K763R and K769R mutations) was almost completely absent. Figure 5 (B), and cross-species sequence alignment analysis showed that these two sites are highly conserved in evolution ( Figure 5 (C).

[0055] To monitor the modification dynamics in real time, specific lactation antibodies Kla763 (with the immunogenic peptide amino acid sequence: CRTAEKTSLSF-k(Lactyl)-SDQ) and Kla769 (with the immunogenic peptide amino acid sequence: CGSSGRTAE-k(Lactyl)-TSLS) targeting these two sites were developed. Their excellent sequence specificity was verified by dot blot and enzyme-linked immunosorbent assay (ELISA). Figure 5 DE), and confirmed the lactation status of TIF1γ at K763 and K769 sites in A549 and H1650 cells at the endogenous level ( Figure 5 (F).

[0056] The specific experimental procedure is as follows:

[0057] (1) Immunogen peptide design and synthesis: Based on the lactation site sequence identified by high-resolution liquid chromatography-tandem mass spectrometry (LC-MS / MS), antigen peptides with specific lactyl modification were designed and artificially synthesized.

[0058] The amino acid sequence of the Kla763 immunogenic peptide is: CRTAEKTSLSF-K(Lactyl)-SDQ (the unmodified immunogenic peptide sequence is shown in SEQ ID NO.3, SEQ ID NO.3: CRTAEKTSLSFKSDQ).

[0059] The amino acid sequence of the Kla769 immunogenic peptide is: CGSSGRTAE-K(Lactyl)-TSLS (the unmodified immunogenic peptide sequence is shown in SEQ ID NO.4, SEQ ID NO.4: CGSSGRTAEKTSLS). Here, Lactyl represents a lactyl group, indicating lactation modification of lysine (K).

[0060] The synthesized modified peptides were conjugated to a carrier protein to serve as a complete antigen. Simultaneously, corresponding wild-type peptides without lactation modification were synthesized for subsequent cross-adsorption and control validation.

[0061] (2) Animal immunization and antibody purification: Experimental animals (New Zealand white rabbits) were immunized multiple times using a standard immunization procedure. After collecting antiserum, it was first subjected to negative phase adsorption (removing pan-antibodies that recognize backbone amino acids) through an affinity chromatography column of wild-type unmodified peptides, and then purified in the normal phase through a peptide affinity chromatography column with lactation modification, finally obtaining highly enriched site-specific lactated antibodies Kla763 and Kla769.

[0062] (3) In vitro specificity verification

[0063] Dot blot: Lactated peptides and unmodified control peptides at different concentration gradients are spotted onto an NC membrane and incubated with purified Kla763 and Kla769 antibodies, respectively, and their recognition signals are detected.

[0064] Enzyme-linked immunosorbent assay (ELISA): Modified and unmodified peptides are coated in microplates, serially diluted specific antibodies are added, and their binding titers and cross-reactivity rates are measured.

[0065] (4) Validation of endogenous modification levels in cells: Total protein was extracted from human lung adenocarcinoma cell lines A549 and H1650. The TIF1γ protein complex was enriched by endogenous immunoprecipitation (IP) using a TIF1γ-specific antibody (brand: CST, catalog number: Cat# 90051, dilution: 1:1000). Subsequently, Western blotting was performed using custom-designed Kla763 and Kla769 antibodies as primary antibodies to detect the true lactation status of endogenous TIF1γ protein at these two sites.

[0066] 3. Experimental Results

[0067] The in vitro sequence recognition specificity is extremely high: the designed Kla763 and Kla769 antibodies only produce strong dose-dependent binding signals to antigenic peptides with corresponding lactation modifications, while showing almost no cross-reactivity signals to wild-type unmodified peptides, confirming their excellent sequence and modification state specificity. Figure 5 (DE).

[0068] Successful capture of endogenous cellular modifications: In A549 and H1650 cells, Kla763 and Kla769 antibodies were able to clearly and accurately identify and detect the K763 and K769 lactation modification bands on the TIF1γ protein. Figure 5 (F).

[0069] In summary, this embodiment successfully prepared highly specific TIF1γ-Kla763 and TIF1γ-Kla769 modified antibodies, providing direct underlying tool support for subsequent screening of targeted drugs antagonizing lactation modification (such as sulfasalazine) and the development of clinical companion diagnostic kits for metastasis risk.

[0070] Finally, this embodiment evaluated whether the specific site modification is directly controlled by lactate homeostasis and acetyltransferase (TIP60, which plays an important role in the occurrence, development and metastasis of tumors. In this embodiment, TIP60 enzyme activity reflects whether tumor metastasis has occurred.) enzyme activity. Wild-type TIF1γ (Flag-TIF1γ-WT) and the lactation dual-site mutant (Flag-TIF1γ-2KR mutant) were overexpressed in 293T cells, respectively. Cells were then treated with lactate (10 mM), the lactate dehydrogenase inhibitor LDHi (10 μM), or co-transfected with Myc-TIP60 plasmid for 24 hours. Immunoprecipitation followed by detection with Kla763 and Kla769 antibodies revealed that in cells overexpressing wild-type TIF1γ, lactation signals at the K763 / K769 sites were significantly enhanced after lactate treatment or TIP60 overexpression, but weakened after LDHi treatment. In contrast, in cells overexpressing the TIF1γ-2KR mutant, lactation signals at both sites remained at extremely low levels and did not change with the above treatments. Figure 5 GI).

[0071] In summary, this embodiment clarifies that the K763 / K769 sites are key "targets" for lactate negative feedback regulation of TIF1γ. Blocking lactation modification at these two sites is the core technical pathway to maintain the stability and subcellular localization of TIF1γ protein and thus reverse cancer metastasis.

[0072] Example 3: Screening and Binding Mode Analysis of Targeted Small Molecule Drugs

[0073] This embodiment utilizes computational structural biology techniques to identify a lead drug capable of physically blocking lactation modification and to resolve its binding conformation with the target protein TIF1γ.

[0074] First, this invention uses AlphaFold3 software to predict and construct three-dimensional structural models of wild-type TIF1γ (TIF1γ-WT) and simulated lactation modification state at K763 / K769 sites (TIF1γ-Kla). Figure 6A). 1000 nanosecond (ns) all-atom molecular dynamics (MD) simulations revealed that lactation modification caused dramatic fluctuations in the root mean square deviation (RMSD) of TIF1γ, with a significant increase in the RMSF value near the K763 / K769 site. This confirms that lactation modification significantly increases the local flexibility of the protein and reduces its conformational stability, thereby inducing pathological degradation. Figure 6 Based on this structural foundation, this embodiment identifies the binding pocket near the K763 / K769 site as the target region. A virtual screening workflow was executed using the ZINC20 DrugBank approved drug library. From 2910 compounds, binding energy scores and conformational fit assessments identified sulfasalazine (SAS), a small molecule drug with the highest binding energy. Figure 6 (C).

[0075] To verify the robustness of sulfasalazine binding to TIF1γ, a 1000 ns MD trajectory analysis was performed. The results showed that in the sulfasalazine-bound state (TIF1γ-WT-loaded state), the RMSD and RMSF fluctuations of the TIF1γ protein significantly converged, indicating that the SAS embedding effectively locked the conformational stability of the lactation pocket. Figure 6 The molecular docking results precisely revealed the interaction details between SAS and TIF1γ: SAS is stably bound within the lactation-sensitive pocket of TIF1γ, forming multiple key hydrogen bonds with key amino acid residues G50, R52, G752, G755, E762, K763, and V773. The binding free energy calculated using the MM / GBSA method reaches -30.5404 kcal / mol. Figure 6 E).

[0076] Subsequently, surface plasmon resonance (SPR) technology was used to confirm, from a biophysical perspective, the direct and dose-dependent binding ability between SAS and TIF1γ protein was demonstrated, and the measured dissociation constant ( Kd The value was 3.76e-05 M, confirming the biological fact that SAS is a TIF1γ specific ligand. Figure 7 (AB).

[0077] In the cell biochemical validation phase, immunoprecipitation experiments revealed that sulfasalazine treatment significantly downregulated the lactation levels of TIF1γ at K763 and K769 sites. Furthermore, when SAS was used in combination with the lactate dehydrogenase inhibitor (LDHi), it exerted a stronger synergistic inhibitory effect by reducing the supply of lactoyl substrate and physically blocking the modification pocket. Figure 7Finally, Western blot analysis revealed that SAS treatment significantly upregulated the protein abundance of TIF1γ and correspondingly reduced the expression of mesenchymal markers such as N-cadherin and vimentin, thereby effectively reversing the epithelial-mesenchymal transition (EMT) process. Figure 7 (D).

[0078] To rule out off-target effects, a reversion experiment was conducted in this embodiment. A549 cells and H1650 cells were treated with either TIF1γ knockdown or TIF1γ re-expression, followed by administration of antitumor steroids (SAS). The effect of SAS on EMT marker expression was detected to assess the drug dependence of SAS, with β-actin used as an internal control. The results confirmed that the anti-metastatic regulatory effect of SAS was essentially lost in cells with TIF1γ knockdown, while its antitumor function was restored after TIF1γ expression was re-expressed, confirming that the pharmacological effect of SAS is highly specific to the molecular target TIF1γ. Figure 7 E).

[0079] Example 4: Validation of the synergistic therapeutic effect of combined drug therapy on lactation modification of TIF1γ

[0080] This embodiment validates the synergistic anti-metastatic effect of the combination of sulfasalazine (SAS) and lactate dehydrogenase inhibitor (LDHi) at a "sub-dose" level and evaluates its clinical translational potential.

[0081] Experimental subjects: human lung adenocarcinoma cell lines A549 and H1650; test drugs: sulfasalazine (SAS) and lactate dehydrogenase inhibitor LDHi (GSK2837808A).

[0082] 1. Experimental Design and Standardized Grouping: For the various cellular and molecular biochemical assays in this embodiment, the following four drug administration groups were uniformly set up:

[0083] Control group (Dimethyl sulfoxide); SAS monotherapy group (SAS 25-200 μM); LDHi monotherapy group (LDHi 1.25-10 μM); combination therapy group (SAS 25-200 μM + LDHi 1.25-10 μM).

[0084] A549 and H1650 cells were seeded in culture dishes. When the cell confluence reached 70%-80%, the corresponding concentration of the drug was added according to the above grouping. The dishes were then placed in a 37°C, 5% CO2 incubator for 24 hours.

[0085] 2. Scratch Healing Assay: A549 and H1650 cells were cultured in 6-well plates until full coverage. Uniform scratches were made using a pipette tip. After washing away detached cells with PBS, the culture medium was replaced with low-serum medium containing the corresponding concentrations of the drugs listed above. The scratch area was recorded at 0 h and 24 h, and the scratch healing rate was calculated.

[0086] 3. Invasion Assay (Transwell Method): Matrigel may or may not be laid on the bottom membrane of the upper chamber of the Transwell chamber. A549 and H1650 cells pretreated with each drug group for 24 hours were collected, resuspended in serum-free medium, and seeded into the upper chamber. The lower chamber was treated with complete medium containing 20% ​​fetal bovine serum as a chemotactic agent. All mediums contained the corresponding concentrations of the drugs mentioned above. After 24 hours of routine culture, cells that had not penetrated the upper chamber were wiped away. Cells that had penetrated the Matrigel and reached the bottom of the lower chamber were fixed, stained with crystal violet, and photographed and counted under a microscope.

[0087] Experimental results: Although single-drug SAS (maximum 200 μM) or single-drug LDHi (maximum 10 μM) could inhibit the invasion and migration of A549 and H1650 cells in a dose-dependent manner, their highest inhibition rate was only maintained at about 35%. Figure 8 (AD). In contrast, the inhibition rate of the 50 μM SAS + 2.5 μM LDHi combination in the combination therapy group reached the level of high-dose single-drug therapy; and when the concentration of LDHi in combination therapy was increased by 5 μM, the 50 μM SAS + 5 μM LDHi combination almost completely blocked the scratch healing and membrane penetration ability of lung adenocarcinoma cells within 24 hours, and the inhibitory efficacy significantly exceeded that of the highest concentration of single-drug therapy group, with an invasion inhibition rate of over 70%, showing a very significant synergistic effect. Figure 8 (EF). Furthermore, based on the intermediate-efficacy principle, the Chou-Talalay method calculated that the synergistic index (CI value) of the combination drug of 50 μM SAS + 5 μM LDHi was distributed between 0.340 and 0.669 (far less than 1, see Table 1-6), which quantitatively confirmed that the two drugs have a very significant low-dose strong synergistic effect in inhibiting cancer cell invasion and metastasis.

[0088] Table 1. CI values ​​of combined drug use in A549 cell invasion assay

[0089]

[0090] Table 2 CI values ​​of combined drugs in A549 cell migration assay

[0091]

[0092] Table 3 CI values ​​of combined drugs in A549 cell scratch healing assay

[0093]

[0094] Table 4. CI values ​​of combined drug use in H1650 cell invasion assay

[0095]

[0096] Table 5 CI values ​​of combined drug use in H1650 cell migration assay

[0097]

[0098] Table 6 CI values ​​of combined drug treatment in H1650 cell scratch healing assay

[0099]

[0100] Example 5: Anti-metastasis value in organisms

[0101] Experimental subject: KSLE mice.

[0102] Operating steps:

[0103] 1. Model Construction and Grouping: 6-8 week old KSLE mice were intratracheally instilled with adenovirus (Ad-Cre, 4×10⁻⁶ mcg) carrying Cre recombinase. 7 PFU / 35μL was used to specifically induce lung tumor formation. Four weeks later, tumor formation was confirmed using in vivo imaging, and patients were randomly assigned to different groups. Figure 9 (AB).

[0104] 2. Sub-dose combination therapy: The SAS dose was 62.5 mg / kg (only 25% of the safe dose for single-drug administration), and the LDHi dose was 3 mg / kg (only 50% of the safe dose for single-drug administration). The control group received an equal volume of normal saline. Administration was performed by gavage every other day for 4 weeks.

[0105] 3. Monitoring of indicators: During the treatment period, small animal in vivo fluorescence imaging was performed weekly to monitor changes in tumor burden. After treatment, lung, liver, spleen and other tissues were dissected and collected for Bouin's staining to count surface tumor nodules, in vitro tissue imaging luminescence detection, and hematoxylin-eosin (H&E) staining to observe micrometastases.

[0106] Experimental Results: After 4 weeks of continuous treatment, in vivo imaging showed strong biofluorescence signals in the lungs of control mice with mediastinal and distal spread, while the fluorescence signal in the combined treatment group (SAS + LDHi) was significantly weakened, and the lesion extent was limited. Subsequent Bouin's staining for gross observation, in vitro chemiluminescence detection, and H&E staining for histological evaluation of mouse lung tissue confirmed that the lungs of the control group were filled with dense micrometastatic nodules, while the number of nodules in the combined treatment group was reduced by more than 80%, the degree of lesion infiltration was significantly reduced, and no distant metastases were observed in organs such as the liver and spleen. Figure 9 The CE (cerebrospinal concentration) of mice was significantly reduced, while the body weight of mice did not decrease significantly after treatment compared with the control group. Figure 9 (F). In addition, pathological examination of stained sections of major organs (lungs, liver, spleen) also confirmed that no significant organ toxicity or organic pathological damage occurred in the combined treatment group.

[0107] This embodiment confirms the superiority of the combination of SAS and LDHi: through the dual mechanism of "source reduction (LDHi reduces lactate donors) + receptor antagonism (SAS competitively occupies the TIF1γ modification pocket)," this regimen can effectively block the vicious cycle of "metabolism-modification-degradation" driven by lactate in cancer while significantly reducing the dosage of individual drugs and avoiding systemic toxicity. This provides a safe, efficient and precise combination strategy for the clinical anti-metastatic treatment of cancer.

[0108] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of sulfasalazine in combination with a lactate dehydrogenase inhibitor in the preparation of a lung adenocarcinoma treatment product, characterized in that, The lactate dehydrogenase inhibitor is GSK2837808A, and the dosage ratio of sulfasalazine to the lactate dehydrogenase inhibitor is 60-70 mg / kg: 2-4 mg / kg.

2. The application according to claim 1, characterized in that, The lung adenocarcinoma treatment product targets and blocks the lactation of lysine residues at positions 763 and 769 of the transcriptional mediator 1γ.

3. The application according to claim 2, characterized in that, The NCBI number for the transcriptional mediator 1γ is NP_056990.

3.

4. The application of sulfasalazine and lactate dehydrogenase inhibitors in the preparation of products that inhibit lung adenocarcinoma metastasis, characterized in that, The lactate dehydrogenase inhibitor is GSK2837808A.

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