Use of caMKII-alpha inhibitors for the preparation of a medicament for the treatment of t-cell lymphoma

Abstract

The application relates to application of a CaMKII-alpha inhibitor in preparation of a medicine for treating T cell lymphoma. The application can obviously down-regulate the expression level of CaMKII-alpha in T cell lymphoma through si-RNA, determines that CaMKII-alpha is a potential treatment target of T cell lymphoma, and identifies Evans blue as an effective CaMKII-alpha inhibitor through high-throughput screening. Experiments show that the Evans blue can obviously inhibit the growth of T cell lymphoma Jurkat cells and H9 cells, and the effect is similar to that of siRNA treatment. In-vivo experiments of Jurkat transplanted tumors also prove that the Evans blue realizes the in-vivo tumor inhibition effect by inhibiting CaMKII-alpha, and reveal a good tumor inhibition effect of the Evans blue on T cell lymphoma.

Description

Technical Field

[0001] This invention belongs to the field of gene therapy drug technology, and relates to a drug for treating T-cell lymphoma, particularly a drug for treating T-cell lymphoma by inhibiting the expression of CaMKII-α in T-cell lymphoma cells. Background Technology

[0002] T-cell lymphoma (TCL) is a malignant disease originating from T lymphocytes. T lymphocytes are responsible for recognizing and clearing pathogens and abnormal cells in the body. When they undergo gene mutations or abnormal proliferation, it can lead to the development and progression of TCL. Common clinical symptoms of TCL include swollen lymph nodes, fever, malaise, and unexplained weight loss.

[0003] Current treatment options for TCL include chemotherapy, radiotherapy, and immunotherapy, with the CHOP chemotherapy regimen (containing cyclophosphamide, doxorubicin, vincristine, and prednisone) widely used as the first-line treatment for various TCL subtypes. While the CHOP regimen is effective, traditional chemotherapy and radiotherapy often come with a range of side effects, such as nausea, vomiting, and hair loss. More importantly, TCL patients are prone to developing resistance to these conventional treatments, highlighting the need to explore new treatment targets and develop more efficient treatment pathways to address this challenge.

[0004] Calmodulin-dependent protein kinase II (CaMKII) family is a class of proteins that depend on calcium ions (Ca). 2+ Protein kinases, including calmodulin (CaMKII) and calcium-μLin, are widely distributed in various organisms. The CaMKII family includes four isoforms: CaMKII-α, CaMKII-β, CaMKII-γ, and CaMKII-δ. CaMKII-α and CaMKII-β isoforms are primarily found in the nervous system and are crucial for regulating various neural functions, especially playing an indispensable role in learning and memory. CaMKII-γ and CaMKII-δ isoforms exhibit a wider tissue distribution. CaMKII-γ is mainly responsible for regulating gene expression and cell proliferation and is closely related to the occurrence and development of cancer; CaMKII-δ dominates in the heart and is essential for maintaining vascular tone and regulating cardiac function.

[0005] The CaMKII family, a key class of protein kinases, plays complex roles in regulating cellular function. CaMKIIs can undergo autophosphorylation, which increases their catalytic activity, enabling them to more effectively phosphorylate downstream substrates. From the functioning of the nervous system to cardiovascular health, and even cell growth and differentiation, the influence of CaMKII family members is ubiquitous, making them crucial targets for exploring physiological and pathological mechanisms.

[0006] siRNA (small interfering RNA) is a short double-stranded RNA molecule, typically 20-25 nucleotides in length. siRNA plays a crucial role in gene silencing and is part of the RNA interference (RNAi) mechanism, which specifically inhibits the expression of target genes. siRNA binds to the RNA-induced silencing complex (RISC) within the cell, directing the complex to recognize and cleave mRNA sequences complementary to the siRNA, thereby preventing these mRNAs from being translated into proteins and downregulating the expression of specific target genes.

[0007] The discovery and development of siRNA technology has greatly advanced research in functional genomics and therapeutics. Scientists can design and synthesize specific siRNAs to target almost any gene of interest, for studying gene function, validating drug targets, and developing new treatments. For example, in cancer research, siRNAs are used to explore the function of oncogenes and assess their potential as therapeutic targets. Furthermore, siRNA therapy is emerging as a new treatment approach for targeting disease targets that are difficult to reach with traditional drugs.

[0008] Evans Blue, with its deep blue appearance, is a water-soluble azo dye. It was first synthesized by the British chemist Henry Evans in 1917, hence its name. Evans Blue has wide applications in biological and medical research. In clinical applications, it can be used to measure blood volume and plasma volume because it rapidly binds to albumin in plasma to form a stable complex, which is then distributed throughout the bloodstream. By measuring the change in dye concentration in the blood after injecting a certain amount of Evans Blue, the blood loss volume can be calculated. Evans Blue is also used in oncology as a staining agent for solid tumors to improve the visualization of tumor boundaries during surgery, and its EPR effect (enhanced permeability and retention effect) allows for selective accumulation and prolonged retention of tumors, facilitating tumor diagnosis and treatment monitoring.

[0009] In addition to the uses mentioned above, Evans blue is also used in fields such as pathology and histochemistry, for example, for staining tissues under a microscope. Overall, Evans blue, as a multifunctional biological dye, plays an important role in scientific research and medical practice. Summary of the Invention

[0010] The purpose of this invention is to provide a CaMKII-α inhibitor and its use in the preparation of a medicament for treating T-cell lymphoma.

[0011] The applicant's previous research found that, compared with normal tissue, the expression of the gene encoding CaMKII-α, CAMK2A, was significantly increased in the pathological tissue of T-cell lymphoma. However, to date, no definitive research has confirmed the relationship between CAMK2A and T-cell lymphoma.

[0012] Based on the above findings, this invention utilizes siRNA as a tool to reduce the expression level of CaMKII-α in order to investigate its role in T-cell lymphoma. By selectively inhibiting CaMKII-α, this invention aims to observe the effects of this inhibition on T-cell lymphoma cell proliferation and signaling pathways, thereby revealing the role of CaMKII-α in the pathogenesis of T-cell lymphoma and evaluating its potential as a therapeutic target.

[0013] Therefore, the present invention first provides the application of CaMKII-α as a drug target in the screening and preparation of drugs for the treatment of T-cell lymphoma.

[0014] This invention identifies CaMKII-α (calcium / calmodulin-dependent protein kinase II-α) as a potential therapeutic target for T-cell lymphoma (TCL) through bioinformatics analysis.

[0015] To further explore the role of CaMKII-α in the pathogenesis of TCL, this invention conducted a series of experimental studies. First, using siRNA technology, the expression level of CaMKII-α was successfully and significantly downregulated in two T-cell lymphoma cell lines, Jurkat and H9. Further cell proliferation assays showed that this downregulation significantly inhibited the in vitro proliferation ability of these two cell lines, indicating that CaMKII-α may promote the growth of TCL cells. Subsequently, through bioinformatics prediction and Western blotting analysis, it was further discovered that the PI3K-AKT signaling pathway may be a key pathway by which CaMKII-α regulates TCL progression. Specifically, after downregulating CaMKII-α expression, the expression levels of related proteins in this pathway changed significantly, suggesting that CaMKII-α may regulate TCL development by affecting the PI3K-AKT signaling pathway.

[0016] Therefore, the above experimental results suggest that CaMKII-α inhibitors can be used as drugs for the treatment of T-cell lymphoma and should be applied in the treatment of T-cell lymphoma. This invention not only reveals the key role of CaMKII-α in the pathogenesis of TCL, but also provides a theoretical basis and practical foundation for the development of novel anticancer drugs targeting CaMKII-α.

[0017] More importantly, this invention uses high-throughput screening to identify a small molecule inhibitor, Evans blue, that can effectively inhibit CaMKII-α protein from the FDA and natural product libraries.

[0018] Therefore, the present invention further provides the application of the small molecule compound Evans blue in the preparation of CaMKII-α inhibitors.

[0019] More specifically, this invention provides the use of the small molecule compound Evans blue in the preparation of a medicament for treating T-cell lymphoma.

[0020] Clearly, the new research findings of this invention regarding the use of Evans blue in the treatment of T-cell lymphoma are significantly different from the traditional use cases of Evans blue.

[0021] This invention demonstrates that Evans blue significantly inhibits the growth of Jurkat and H9 T-cell lymphoma cells, and also significantly inhibits the growth of Jurkat xenografts. Tumor tissue treated with Evans blue was significantly smaller than that in the control group, and the changes in PI3K-AKT signaling pathway-related proteins in the tumor tissue were consistent with in vitro experiments. This indicates that Evans blue achieves its in vivo tumor-suppressing effect by downregulating the expression of CaMKII-α in T-cell lymphoma cells and inhibiting the proliferation of T-cell lymphoma cells.

[0022] This new discovery suggests that Evanslan may have a therapeutic effect on T-cell lymphoma by inhibiting CaMKII-α, which is completely different from the traditional application of Evanslan. It expands the application of Evanslan from a simple diagnostic tool to a potential therapeutic agent, especially for specific types of cancer such as TCL. This research not only provides new insights into the biological function of Evanslan but also paves the way for the development of new anticancer drugs.

[0023] Furthermore, based on the above experimental studies, the present invention provides a drug for treating T-cell lymphoma, wherein the drug contains the above-mentioned CaMKII-α inhibitor, which inhibits the proliferation of T-cell lymphoma cells by downregulating the expression of CaMKII-α in T-cell lymphoma cells, as the active pharmaceutical ingredient.

[0024] Furthermore, the active pharmaceutical ingredient contained in the medicament for treating T-cell lymphoma provided by the present invention is preferably the small molecule compound Evans blue.

[0025] The experiments of this invention demonstrate that the expression of CaMKII-α can promote the growth and proliferation of T-cell lymphoma, while Evans blue exerts its in vivo tumor-suppressing effect by inhibiting CaMKII-α. Similar to the results of siRNA treatment, it inhibits the proliferation of T-cell lymphoma cells by regulating the protein expression in the PI3K-AKT signaling pathway in cells. Attached Figure Description

[0026] Figure 1 This reflects the differences in expression of genes in the CAMKII family between healthy tissues and tissues from patients with T-cell lymphoma.

[0027] Figure 2 This refers to the change in the expression level of CaMKII-α protein in the transfected Jurkat and H9 T-cell lymphoma cell lines compared to the control group.

[0028] Figure 3 This refers to the proliferative activity of Jurkat and H9 T-cell lymphoma cell lines in each transfection group compared to the control group.

[0029] Figure 4 Compared with healthy tissue, the tissues of patients with T-cell lymphoma show an enrichment of related signaling pathways.

[0030] Figure 5 This refers to the changes in the expression levels of proteins related to the PI3K-AKT signaling pathway in the Jurkat and H9 T-cell lymphoma cell lines of each transfection group compared with the control group.

[0031] Figure 6 The structural formula of Evans blue and its IC50 for CaMKII-α protein. 50 value.

[0032] Figure 7 Evans blue is the IC50 of Jurkat and H9 cell lines of T-cell lymphoma. 50 value.

[0033] Figure 8 This refers to the changes in the expression levels of proteins related to the PI3K-AKT signaling pathway in Jurkat and H9 T-cell lymphoma cell lines after Evans blue treatment, compared with the control group.

[0034] Figure 9 This is a comparison of the changes in mice in the treatment group and the control group, and the final tumor size.

[0035] Figure 10This refers to the changes in the expression levels of PI3K-AKT signaling pathway-related proteins in the tumors of mice in the treatment group compared to the control group. Implementation

[0036] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present invention, so that those skilled in the art can better understand and utilize the present invention, and are not intended to limit the scope of protection of the present invention.

[0037] Unless otherwise specified, the production processes, experimental methods, or testing methods involved in the embodiments of this invention are all conventional methods in the prior art, and their names and / or abbreviations are all conventional names in the field, which are very clear and distinct in the relevant application areas. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment, and implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0038] The various instruments, equipment, raw materials or reagents used in the embodiments of this invention are not subject to any special restrictions on their source. They are all conventional products that can be purchased through regular commercial channels and can be prepared according to conventional methods known to those skilled in the art. Example

[0039] Example 1

[0040] This embodiment uses bioinformatics to analyze the expression of the CAMKII family in healthy tissue samples and T-cell lymphoma patient tissue samples, and uses si-RNA to target and downregulate the expression of CaMKII-α.

[0041] I. Experimental Materials

[0042] Jurkat and H9 cell lines for T-cell lymphoma were purchased from Nanjing Herbal Source Biotechnology Co., Ltd.

[0043] The specific CAMK2A si-RNA was synthesized by Guangzhou Ruibo Biotechnology Co., Ltd., and its specific gene sequence is as follows:

[0044] Chain of Justice: 5'-CACCACCAUUGAGGACGAA-3';

[0045] Antonym chain: 5'-UUCGUCCUCAAUGGUGGUG-3'.

[0046] II. Experimental Methods

[0047] 1. Bioinformatics analysis of the expression of the CAMKII family in healthy tissue samples and tissue samples from T-cell lymphoma patients.

[0048] This study used R4.3.0 for statistical analysis and examined the T-cell lymphoma dataset (GSE45712) from the Gene Expression Comprehensive Database (GEO), which includes microarray data from the control group (CON) and the tumor group (Tumor).

[0049] Initially, the "normalized intermediate array" function in the "limma" package was used to standardize sequencing depth across samples, mitigating technical biases that could affect subsequent analyses. Subsequently, four members of the CAMKII family—CAMK2A, CAMK2B, CAMK2D, and CAMK2G—were examined, and the "characteristic state map" function in the "scprep" package was used to assess differential expression patterns of these genes in different samples, revealing their potential biological significance.

[0050] 2. Cell culture and transfection

[0051] Jurkat and H9 T-cell lymphoma cell lines were cultured using RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies according to standard methods.

[0052] Jurkat cells and H9 cells were divided into siRNA transfection group and blank control group, respectively. Transfection was performed in six-well cell culture plates when the cell binding rate was about 60-70%.

[0053] Jurkat cell lines were first seeded in OPTI-reduced serum medium. After 0.5-3 hours, 100 nM si-RNA and Lipofectamine 3000 transfection reagent were added to the wells of the si-RNA transfection group. The transfection process was strictly performed according to the Lipofectamine 3000 instructions. The medium was changed 24 hours after transfection. The same procedure was performed on the blank control group, but without the addition of si-RNA.

[0054] H9 cell lines were first plated using standard culture medium. 100 nM si-RNA and CALNPTMRNAi in vitro transfection reagent were added to the wells of the si-RNA transfection group. The transfection process was strictly performed according to the CALNPTMRNAi in vitro instructions. The medium was changed 24 hours after transfection. The same procedure was performed on the blank control group, but without the addition of si-RNA.

[0055] 3. Western blotting method for detecting CaMKII-α expression level

[0056] Cells were harvested 72 h after transfection, washed three times with PBS buffer, and the four groups of cells were lysed on ice with RIPA lysis buffer. The supernatant was collected by centrifugation, and the protein content of the cell samples was measured using a BCA kit.

[0057] Using GAPDH as an internal control, cell samples were loaded with 20 μg of protein and subjected to SDS-PAGE gel electrophoresis; proteins were transferred to PVDF membranes by wet transfer method and blocked with protein-free rapid blocking solution.

[0058] The PVDF membrane was reacted overnight at 4°C with CaMKII-α primary antibody dilution buffer and GAPDH primary antibody dilution buffer, followed by three TBST washes for 10 min each. The membrane was then incubated with HRP-labeled secondary antibody at room temperature for 2 h, followed by three TBST washes for 10 min each. ECL chromogenic buffer was added, and the membrane was photographed using an automated gel analyzer. The relative expression levels of the corresponding proteins were obtained by dividing the corresponding protein band gray values ​​by the internal control GAPDH gray value.

[0059] 4. Cell proliferation activity detection

[0060] CellTiter-Glo ® The proliferation activity of transfected cells was detected by luminescence assay, and the assays were performed within 1-6 days after transfection.

[0061] Cells were seeded in 96-well plates, with 5000 cells and 100 μL of culture medium added to each well. After adding 100 μL of CellTiter, the plates were shaken for 10 min to allow for full reaction. The luminescence value of each well was measured using a microplate reader. Each group was repeated 3 times.

[0062] Relative proliferation activity (%) = luminescence value of transfected group / luminescence value of blank control group × 100%.

[0063] III. Experimental Results

[0064] 1. Bioinformatics Analysis Results

[0065] The results showed that, compared with healthy tissue samples, CAMK2A expression was significantly upregulated in tissue samples from T-cell lymphoma patients. Figure 1 As shown.

[0066] 2. Comparison of CaMKII-α protein expression levels among different groups after transfection

[0067] Compared with the blank control group, the expression level of CaMKII-α protein in Jurkat cells and H9 cells transfected with si-RNA was significantly downregulated, as shown in the results. Figure 2 As shown.

[0068] 3. Results of cell proliferation activity assay

[0069] Compared with the blank control group, the proliferation of Jurkat cells and H9 cells transfected with si-RNA was significantly slower, as shown in the following results. Figure 3 As shown.

[0070] Cancer is a highly heterogeneous disease; even within the same type of cancer, tumor cells can exhibit significant differences between patients. Using two or more cell lines can help validate the consistency of the effects of drugs, gene editing, or other interventions. If the same results are observed in multiple cell lines, the results are considered more reliable.

[0071] To verify the reliability of the experimental results, this invention selected the Jurkat and H9 T-cell lymphoma cell lines for research. The results showed that siRNA significantly downregulated the expression level of CaMKII-α in both Jurkat and H9 T-cell lymphoma cells. Further cell proliferation assays indicated that downregulating CaMKII-α expression with siRNA significantly inhibited the in vitro proliferation of Jurkat and H9 cells. This result suggests that CaMKII-α may be an important molecular target regulating the proliferation of T-cell lymphoma cells.

[0072] Example 2

[0073] This embodiment utilizes bioinformatics analysis to enrich signaling pathways in tissue samples from T-cell lymphoma patients. By using siRNA to target and downregulate the expression of CaMKII-α in T-cell lymphoma cell lines, changes in signaling pathway-related proteins were detected.

[0074] I. Experimental Methods

[0075] 1. Bioinformatics analysis of signaling pathways significantly enriched in tissues of T-cell lymphoma patients

[0076] This study used R4.3.0 for statistical analysis and examined the T-cell lymphoma dataset (GSE45712) from the Gene Expression Comprehensive Database (GEO).

[0077] An adjusted p-value less than 0.05 was used to control for false positives in multiple comparisons. A log-fold change (logFC) greater than 2 indicated that the gene expression level in tumor samples was at least upregulated by 2-fold (or downregulated by 0.5-fold) compared to normal samples. Based on these stringent criteria, this invention identified a total of 1789 genes that were significantly upregulated in tumor samples.

[0078] To further explore the functions of these differentially expressed genes, this invention used the R package "clusterProfiler" to perform KEGG pathway enrichment analysis. Differentially expressed genes were mapped to a KEGG pathway database, and the enrichment significance of each pathway was calculated. KEGG entries with a p-value < 0.05 were selected as significantly enriched pathways. The selected significantly enriched pathways were exported as an Excel file for subsequent analysis and visualization.

[0079] To visually represent the results of KEGG enrichment analysis, this invention uses the dotplot function to draw a dot plot (or bubble plot). The plot uses the size and color of the dots to represent the number of genes in the pathway and the enrichment significance (-log10(p-value)), respectively, thereby helping to identify signaling pathways closely related to T-cell lymphoma.

[0080] 2. Cell culture and transfection

[0081] Jurkat and H9 T-cell lymphoma cell lines were cultured using RPMI 1640 medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibodies according to standard methods.

[0082] Jurkat cells and H9 cells were divided into siRNA transfection group and blank control group, respectively. Transfection was performed in six-well cell culture plates when the cell binding rate was about 60-70%.

[0083] Jurkat cell lines were first seeded in OPTI-reduced serum medium. After 0.5-3 hours, 100 nM si-RNA and Lipofectamine 3000 transfection reagent were added to the wells of the si-RNA transfection group. The transfection process was strictly performed according to the Lipofectamine 3000 instructions. The medium was changed 24 hours after transfection. The same procedure was performed on the blank control group, but without the addition of si-RNA.

[0084] H9 cell lines were first plated using standard culture medium. 100 nM si-RNA and CALNPTMRNAi in vitro transfection reagent were added to the wells of the si-RNA transfection group. The transfection process was strictly performed according to the CALNPTMRNAi in vitro instructions. The medium was changed 24 hours after transfection. The same procedure was performed on the blank control group, but without the addition of si-RNA.

[0085] 3. Western blotting was used to detect changes in the expression levels of related proteins in the PI3K-AKT signaling pathway.

[0086] Cells were harvested 72 h after transfection, washed three times with PBS buffer, and the four groups of cells were lysed on ice with RIPA lysis buffer. The supernatant was collected by centrifugation, and the protein content of the cell samples was measured using a BCA kit.

[0087] Using GAPDH as an internal control, cell samples were loaded with 20 μg of protein and subjected to SDS-PAGE gel electrophoresis; proteins were transferred to PVDF membranes by wet transfer method and blocked with protein-free rapid blocking solution.

[0088] The PVDF membrane was reacted overnight at 4°C with dilution buffers for PI3K, AKT, p-PI3K, p-AKT, and GAPDH primary antibodies, followed by three TBST washes for 10 min each. The membrane was then incubated with HRP-labeled secondary antibody at room temperature for 2 h, followed by three TBST washes for 10 min each. ECL chromogenic buffer was added, and the membrane was photographed using an automated gel analyzer. The relative expression levels of the corresponding proteins were obtained by dividing the corresponding protein band gray values ​​by the internal control GAPDH gray value.

[0089] III. Experimental Results

[0090] 1. Bioinformatics Analysis Results

[0091] Bioinformatics analysis revealed a significant enrichment of the PI3K-AKT signaling pathway in tissues of T-cell lymphoma patients compared to healthy tissues. The results are as follows: Figure 4 As shown.

[0092] 2. Changes in the expression levels of PI3K-AKT signaling pathway-related proteins in each group after transfection.

[0093] Compared with the blank control group, the expression level of CaMKII-α protein in Jurkat cells and H9 cells transfected with si-RNA was significantly downregulated. The expression levels of PI3K and AKT proteins in the PI3K-AKT signaling pathway did not change significantly, while the expression levels of p-PI3K and p-AKT proteins were downregulated. The results are as follows: Figure 5 As shown.

[0094] The above results indicate that both the CAMK2A and PI3K-AKT signaling pathways are significantly overexpressed in tissues of T-cell lymphoma patients. Using siRNA technology, the expression level of CaMKII-α in T-cell lymphoma cell lines (including Juekat cells and H9 cells) was effectively downregulated, while the phosphorylation level of related proteins in the PI3K-AKT signaling pathway was significantly reduced. This finding suggests that CAMK2A may play an important role in regulating the activity of the PI3K-AKT signaling pathway, and its mechanism may be through directly or indirectly affecting the phosphorylation status of these proteins, thereby participating in the occurrence and development of T-cell lymphoma.

[0095] Example 3

[0096] This embodiment utilizes CaMKII-α as a drug target to screen small molecule compounds that have the effect of targeting and inhibiting CaMKII-α.

[0097] I. Experimental Materials

[0098] The ADP-Glo™ kinase assay kit was purchased from Promega, Inc., USA.

[0099] FDA compound library and natural product library, purchased from MCE.

[0100] Evans Blue, CAS No.: 314-13-6, purchased from MCE.

[0101] II. Experimental Methods

[0102] 1. Screening of CaMKII-α inhibitor drugs

[0103] This invention first constructs a high-throughput screening system, employing luminescence immunoassay to detect the effect of drugs on the activity of human CaMKII-α recombinant protein phosphotransferase, in order to screen for potential inhibitors that can effectively inhibit CaMKII-α. In this system, when CaMKII-α is activated, it can catalyze the phosphorylation reaction of the substrate and convert ATP into ADP.

[0104] In this experiment, autocamtide-3 was selected as the peptide substrate for CaMKII-α, and its phosphorylation process is closely linked to the conversion of ATP to ADP.

[0105] To quantitatively assess kinase activity, this invention utilizes the ADP-Glo™ kinase assay kit. This kit indirectly reflects the activity of CaMKII-α by detecting the level of ADP (adenosine diphosphate) generated during the kinase reaction.

[0106] Its working principle is as follows: First, chemical reagents are used to convert ADP in the system into a stable chemical form, thereby preventing further metabolism or degradation of ADP; then, a specific enzyme mixture is added to quench unconsumed ATP and convert the remaining ADP into a product that can generate a light-emitting signal; finally, the kinase activity is accurately quantified by detecting the light intensity.

[0107] The total volume of the reaction system was 10 μL. The reaction buffer consisted of: 25 mmol / L Tris-HCl (pH 7.5), 0.2 mmol / L calcium chloride, 10 mmol / L magnesium chloride, 0.035 mmol / L ATP, 200 μmol / L autocamtide-3, 30 nmol / L calmodulin (CaM), and 0.1 mg / mL bovine serum albumin (BSA).

[0108] The specific experimental steps are as follows: At the beginning of the experiment, 1 μL of the drug to be tested was added to each well, followed by 2 μL (2 ng / μL) of human CaMKII-α recombinant protein, and incubated for 10 min to ensure sufficient interaction between the drug and protein. Next, reaction buffer was added, and the kinase reaction was allowed to run at room temperature (25-27℃) for 30 min. Finally, the reaction system was treated according to the ADP-Glo™ kinase assay kit instructions to stop the kinase reaction and remove unreacted ATP. Through subsequent enzymatic reactions, ADP was converted to ATP, and the luminescent reaction between ATP and luciferin was catalyzed by luciferase, generating a light signal proportional to the amount of ADP, thereby reflecting the phosphotransferase activity of CaMKII-α.

[0109] During the experiment, all operations were strictly performed in accordance with the ADP-Glo™ kinase assay kit instructions. Finally, the luminescence values ​​of each well were measured using a microplate reader, and the potential inhibitory capacity of the compounds was evaluated by quantitatively analyzing their inhibitory effects on protein kinase activity.

[0110] Drug inhibition rate (%) = (luminescence value of drug group - luminescence value of negative control group) / (luminescence value of positive control group - luminescence value of negative control group) × 100%.

[0111] Based on the aforementioned high-throughput screening system, a large-scale screening of 5897 compounds in the MCE's FDA compound library and natural product library was conducted, initially identifying compounds with an inhibition rate greater than 80% against CaMKII-α. Subsequently, this invention further evaluated the effects of different compounds at a series of concentration gradients on CaMKII-α protein activity using this system, and calculated the IC50 of the drug against CaMKII-α protein based on dose-response curve fitting. 50 Values ​​were also measured. The activity of these compounds against other members of the CaMKII family of proteins was also examined to assess their selectivity.

[0112] Based on the comprehensive analysis described above, this invention ultimately identified a compound with the best inhibitory effect and the highest selectivity as a potential inhibitor of CaMKII-α. This systematic screening and validation process ensures the high efficiency and specificity of the selected compound in targeting CaMKII-α.

[0113] 2. Cellular IC 50 Detection

[0114] After confirming that the drug exhibits inhibitory and selective activity against CaMKII-α protein, the next step is to evaluate whether the drug can effectively inhibit cell activity. For this purpose, this invention selected 5.0 × 10⁻⁶... 3One T-cell lymphoma cell line (Jurkat or H9) was seeded into a 96-well plate and treated with a series of drug concentration gradients (10 nM to 1 mM). The total volume per well was set at 100 μL, and the cells were then cultured for 48 h.

[0115] After cultivation, CellTiter-Glo was used. ® Cell viability in each group was detected using a luminescence assay. The specific procedure was as follows: 100 μL of CellTiter-Glo was added to each well. ® The reagents were prepared, and the 96-well plate was shaken for 10 minutes to ensure sufficient reaction between the reagents and cells. Finally, the luminescence values ​​of cells at different drug concentrations were measured using a microplate reader. Each experiment was repeated three times to improve data reliability. The IC50 value of the drug against T-cell lymphoma (Jurkat and H9 cell lines) was calculated based on dose-response curve fitting. 50 The value reflects the overall inhibitory effect of the drug at the cellular level.

[0116] III. Experimental Results

[0117] 1. Drug screening results

[0118] We screened 5,897 compounds from the MCE's FDA compound library and natural product library, selecting compounds with inhibition rates greater than 80%. We then performed activity and selectivity verification on these compounds and selected Evans blue, the compound with the best inhibition rate and selectivity, as the inhibitor of CaMKII-α.

[0119] Chemical structure of Evans blue and its IC50 of CaMKII-α protein 50 Value results are as follows Figure 6 As shown.

[0120] 2. Cell proliferation activity test results

[0121] Compared with the blank control group, cell proliferation was reduced in the Evans blue-treated group in a concentration-dependent manner. Based on the effects of different concentrations of Evans blue on cells, the IC50 of the cells was obtained. 50 Value results are as follows Figure 7 As shown.

[0122] The above results demonstrate that Evans blue, at a low concentration of approximately 197.1 nM, can significantly inhibit the activity of CaMKII-α protein, exhibiting excellent inhibitory effect and high selectivity against the target protein. Simultaneously, at the cellular level, Evans blue significantly inhibited the proliferation of T-cell lymphoma cell lines (H9 and Jurkat cell lines) at concentrations of 3.241 μM and 3.288 μM, showing a clear concentration-dependent effect, fully demonstrating its excellent inhibitory ability against tumor cells.

[0123] In conclusion, Evans blue not only exhibits highly effective inhibitory effects on CaMKII-α protein, but also demonstrates good anti-tumor activity in cell experiments, providing strong evidence for its potential role as a CaMKII-α inhibitor.

[0124] Example 4

[0125] This embodiment verifies the effect of Evans blue, a small molecule compound that targets and inhibits CaMKII-α, on T-cell lymphoma through cell experiments.

[0126] I. Experimental Materials

[0127] p-CaMKII-α primary antibody was purchased from Cell Signaling Technology, and HRP-labeled secondary antibody was purchased from Abcam.

[0128] Other experimental materials are the same as in Examples 1 and 2.

[0129] II. Experimental Methods

[0130] 1. Cell drug delivery treatment

[0131] After learning that Evans blue has an IC50 effect on T-cell lymphoma cells 50 After the value was obtained, the present invention treated the cells with the corresponding concentration of Evans blue for 24 hours.

[0132] 2. Western blotting was used to detect changes in the expression levels of proteins related to the PI3K-AKT signaling pathway in T-cell lymphoma cells after drug administration.

[0133] After culturing for 48 hours following drug administration, cells were harvested, washed three times with PBS buffer, and each group of cells was lysed on ice using RIPA lysis buffer. The supernatant was collected by centrifugation, and the protein content of the cell samples was measured using a BCA kit.

[0134] Using GAPDH as an internal control, cell samples were loaded with 20 μg of protein and subjected to SDS-PAGE gel electrophoresis; proteins were transferred to PVDF membranes by wet transfer method and blocked with protein-free rapid blocking solution.

[0135] The PVDF membrane was reacted overnight at 4°C with CaMKII-α, p-CaMKII-α, PI3K, AKT, p-PI3K, p-AKT primary antibody dilution buffer and GAPDH primary antibody dilution buffer. The membrane was washed three times with TBST for 10 min each time. It was then incubated with HRP-labeled secondary antibody at room temperature for 2 h, followed by three TBST washes for 10 min each time. ECL chromogenic buffer was added, and the membrane was photographed using an automated gel analyzer. The relative expression levels of the corresponding proteins were obtained by dividing the corresponding protein band gray value by the internal control GAPDH gray value.

[0136] III. Experimental Results

[0137] Figure 8 The changes in the expression levels of proteins related to the PI3K-AKT signaling pathway in each group after drug treatment are presented.

[0138] Compared with the blank control group, the expression level of CaMKII-α protein in cells of the Evans blue-treated group did not change significantly, while the expression level of p-CaMKII-α (phosphorylated CaMKII-α) protein decreased. The expression levels of PI3K and AKT proteins in the PI3K-AKT signaling pathway did not change significantly, while the expression levels of p-PI3K and p-AKT proteins were downregulated.

[0139] The above results indicate that drug treatment may affect the PI3K-AKT signaling pathway by inhibiting the autophosphorylation of CAMK2A. This result is consistent with the results observed in siRNA experiments, further verifying the key role of CAMK2A in this signaling pathway.

[0140] Example 5

[0141] This embodiment uses an in vivo experiment in tumor-bearing mice to verify the in vivo tumor-suppressing effect of Evans blue, a small molecule compound that targets and inhibits CaMKII-α, on T-cell lymphoma.

[0142] I. Experimental Materials

[0143] SPF-grade NOD / SCID mice, 7 weeks old and female, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. All mice were housed in an environment with a temperature of (22±2)℃, humidity of (60±5)℃, and 12 hours of alternating light and dark conditions.

[0144] II. Experimental Methods

[0145] 1. Establishment, grouping, and administration of animal models

[0146] Jurkat T-cell lymphoma cells in logarithmic growth phase were collected, prepared into a single-cell suspension with PBS, and counted using trypan blue staining to ensure that the viable cell count was above 95%. The cell density was then adjusted to 5 × 10⁶ cells / year. 7 / mL.

[0147] 0.2 mL of cell suspension was inoculated into the right abdomen of each NOD-SCID mouse, and the mouse's weight was measured before surgery. The mice were observed daily after inoculation. Once a visible xenograft appeared, the xenograft was measured with calipers. The xenograft was considered complete when its volume reached 100 mm². 3 Around 100 mice, tumor-bearing mice were randomly divided into a control group and an Evans blue group based on the principle of balanced tumor volume and body weight.

[0148] The Evans blue group received a dose of 50 mg / kg via tail vein injection, three times a week. The blank control group received an equal volume of normal saline.

[0149] One day after the drug treatment ended, the mice were euthanized by cervical dislocation, photographed, and the shape and size of the mass were observed and recorded. The mass was dissected, weighed, and stored at -80℃ for later use.

[0150] 2. Tumor inhibition rate measurement

[0151] Measure the maximum longitudinal diameter (a) and maximum transverse diameter (b) of the tumor once using vernier calipers. The tumor volume is calculated using the formula: Tumor Volume = 0.5 × a × b. 2 Calculate the tumor volume, and then calculate the in vivo tumor inhibition rate of the compound using the following formula:

[0152] Tumor inhibition rate (%) = (Tumor volume of control group - Tumor volume of treatment group) / Tumor volume of control group × 100%.

[0153] 3. Western blot analysis of changes in the expression levels of proteins related to the PI3K-AKT signaling pathway in mouse tumors.

[0154] Tumor tissue was obtained, ground, and total protein was extracted from the tumor tissue specimen. The tissue was lysed on ice for 30 min using RIPA lysis buffer, and the supernatant was collected by centrifugation. The protein content of the tumor tissue specimen was measured using a BCA kit.

[0155] Using GAPDH as an internal control, tissue samples were loaded with 20 μg of protein and subjected to SDS-PAGE gel electrophoresis; the protein was transferred to a PVDF membrane by wet transfer method and blocked with a protein-free rapid blocking solution.

[0156] The PVDF membrane was reacted overnight at 4°C with CaMKII-α, p-CaMKII-α, PI3K, AKT, p-PI3K, p-AKT primary antibody dilution buffer and GAPDH primary antibody dilution buffer. The membrane was washed three times with TBST for 10 min each time. It was then incubated with HRP-labeled secondary antibody at room temperature for 2 h, followed by three TBST washes for 10 min each time. ECL chromogenic buffer was added, and the membrane was photographed using an automated gel analyzer. The relative expression levels of the corresponding proteins were obtained by dividing the corresponding protein band gray value by the internal control GAPDH gray value.

[0157] III. Experimental Results

[0158] 1. The effect of compounds on tumor growth

[0159] After a period of experimental observation, compared with the control group, the tumor volume of mice in the Evans blue group was significantly smaller, but the body weight of mice in both groups did not change significantly. This result indicates that Evans blue has a significant tumor-suppressing effect in vivo and does not produce obvious systemic toxicity in mice.

[0160] The comparison results of the final tumor size in mice between the Evans blue group and the control group are as follows: Figure 9 As shown.

[0161] 2. Western blot analysis of changes in the expression levels of proteins related to the PI3K-AKT signaling pathway in mouse tumors.

[0162] Compared with the control group, the expression level of CaMKII-α protein in tumors of mice in the Evans blue group did not change significantly, while the expression level of p-CaMKII-α protein decreased. In the PI3K-AKT signaling pathway, the expression levels of PI3K and AKT proteins did not change significantly, while the expression levels of p-PI3K and p-AKT proteins were downregulated. The results are as follows: Figure 10 As shown.

[0163] The above results indicate that Evans blue has a significant inhibitory effect on the growth of Jurkat T-cell lymphoma xenografts. The tumor tissue treated with Evans blue was significantly smaller than that in the control group, indicating that the expression of CaMKII-α can promote the growth and proliferation of T-cell lymphomas. Evans blue achieves its tumor-suppressing effect in vivo by inhibiting CaMKII-α.

[0164] The above embodiments of the present invention do not describe all details exhaustively, nor do they limit the present invention to the embodiments described above. Various changes, modifications, substitutions, and variations made by those skilled in the art to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. Application of the small molecule compound Evans blue in the preparation of drugs for treating T-cell lymphoma.

2. The application according to claim 1, characterized in that it is a small molecule. The compound Evans blue inhibits the proliferation of T-cell lymphoma cells by downregulating the expression of CaMKII-α in T-cell lymphoma cells.

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