Application of IGF2BP2 inhibitors in the preparation of drugs for treating T-ALL

Abstract

The present invention discloses the use of an IGF2BP2 inhibitor in the preparation of a drug for treating T-ALL, belonging to the fields of molecular biology and medicine. The invention clarifies the role of IGF2BP2 in acute T-lymphocytic leukemia, screens for drugs that specifically target and bind to IGF2BP2, and verifies their killing effect on acute T-lymphocytic leukemia cells, providing new therapeutic ideas and drugs for further improving the treatment of acute T-lymphocytic leukemia.

Description

Technical Field

[0001] The present invention belongs to the field of molecular biology and medical technology, and relates to the application of IGF2BP2 as a target in the treatment of T-ALL, and more specifically to the role played by the IGF2BP2 gene in acute T-lymphocytic leukemia, and the application of drugs that specifically target and bind to IGF2BP2 in the treatment of acute T-lymphocytic leukemia. Background Art

[0002] The information in this background section is only intended to enhance understanding of the general background of the invention and is not necessarily to be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Acute T-cell lymphoblastic leukemia (T-ALL) is a hematopoietic malignancy that poses a serious threat to human health. Recent advances in T-ALL treatment have enabled most patients to achieve complete remission (CR), but relapse rates remain high, and long-term survival rates are only 25%-50%. Therefore, further elucidation of the pathogenesis of T-ALL, assessment of risk factors, and identification of new therapeutic targets are urgently needed. Summary of the Invention

[0004] Based on the problems existing in the above-mentioned prior art, the inventors conducted research and analysis from the perspective of the application of IGF2BP2 as a target in the treatment of T-ALL. The present invention clarifies the role of IGF2BP2 in acute T-lymphocytic leukemia, screens drugs that specifically target and bind to IGF2BP2, and verifies their killing effect on acute T-lymphocytic leukemia cells, providing new treatment ideas and drugs for further improving the treatment effect of acute T-lymphocytic leukemia.

[0005] In view of the above research findings, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides use of a reagent for detecting IGF2BP2 gene expression in the preparation of a diagnostic reagent for acute T-lymphocytic leukemia.

[0007] In a second aspect, the present invention provides use of a substance that specifically targets and binds to IGF2BP2 in the preparation of a product for treating acute T lymphocytic leukemia.

[0008] The third aspect of the present invention provides the use of JX5 as an IGF2BP2 targeted inhibitor.

[0009] The present invention has achieved the following technical effects:

[0010] The research results of the present invention clarify the role and mechanism of IGF2BP2 in acute T-lymphocytic leukemia. According to the research results of the present invention, inhibiting IGF2BP2 can achieve a therapeutic effect on acute T-lymphocytic leukemia and inhibit tumor growth.

[0011] The research results of the present invention screened specific targeted binding drugs for IGF2BP2, which is beneficial to the development of clinical therapeutic drugs for acute T lymphocytic leukemia and has important clinical significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The drawings and descriptions of the drawings in the present invention specification are intended to provide a further understanding of the present invention and do not constitute an improper limitation of the present invention.

[0013] Figure 1 The expression of IGF2BP2 in bone marrow cells of T-ALL patients and healthy controls described in Example 1 is shown.

[0014] Figure 2 The biological function of cells after down-regulating the expression of IGF2BP2 in T-ALL cell lines as described in Example 1; wherein, Figure 2 A is the verification of IGF2BP2 expression after downregulation, Figure 2 B is cell proliferation, Figure 2 C is the cell cycle, Figure 2 D stands for cell apoptosis.

[0015] Figure 3 The leukemic effect of IGF2BP2 was confirmed in the T-ALL xenograft mouse model described in Example 1; wherein Figure 3 A is the experimental flow chart, Figure 3 B is the verification of down-regulation of IGF2BP2 expression in cells, Figure 3 C is the survival curve, Figure 3 D is a typical photo of bone marrow and spleen. Figure 3 E is a typical HE staining picture of bone marrow and spleen. Figure 3 F is the expression ratio of human CD45 in peripheral blood, bone marrow, and spleen.

[0016] Figure 4 Schematic diagram of the screening process for IGF2BP2 subunit inhibitors.

[0017] Figure 5 is a small molecule inhibitor of IGF2BP2 obtained by screening; Figure 5 A is the chemical structural formula of compound JX5, Figure 5 B is the molecular docking of compound JX5 and IGF2BP2, Figure 5 C is the structural formula of a derivative of JX5 that may have IGF2BP2 inhibitory activity.

[0018] Figure 6 The case where compound JX5 inhibits the activity of IGF2BP2; wherein, Figure 6 A is an in vitro fluorescence titration experiment confirming the interaction between JX5 and IGF2BP2. Figure 6 B shows the effect of JX5 on the proliferation of HPB-ALL (low expression of IGF2BP2) and Jurkat (high expression of IGF2BP2) cells.

[0019] Figure 7 The effect of compound JX5 on the biological functions of T-ALL cell lines; Figure 7 A is the effect of gradient concentrations of compound JX5 on the proliferation function of IGF2BP2 overexpression / knockout cells and respective control cells, Figure 7 B is the effect of compound JX5 on the proliferation function of IGF2BP2 overexpression / knockout cells and respective control cells with gradient time, Figure 7 C shows the effect of compound JX5 on apoptosis of T-ALL cell lines.

[0020] Figure 8 The effect of compound JX5 on the mRNA level of IGF2BP2 in T-ALL cells. DETAILED DESCRIPTION

[0021] The following detailed description is illustrative and is intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0022] As described in the background art, there is an urgent need to further elucidate the pathogenesis of T-ALLL, assess high-risk factors, and identify new therapeutic targets. The present invention aims to clarify the role of IGF2BP2 in acute T-lymphoblastic leukemia, screen for drugs that specifically target and bind IGF2BP2, and verify their killing effect on acute T-lymphoblastic leukemia cells, thereby providing new treatment ideas and drugs to further improve the treatment effect of acute T-lymphoblastic leukemia.

[0023] Insulin-like growth factor 2 messenger RNA-binding protein 2 (IGF2BP2) is a highly conserved single-stranded RNA-binding protein in eukaryotes that plays a crucial role in embryogenesis. IGF2BP2 regulates gene expression by intervening in all stages of mRNA metabolism, including transcription, 5' end capping, pre-mRNA splicing, 3' end processing, nuclear export, transport, translation, and stability. As an RNA-binding protein, IGF2BP2 likely has hundreds or even thousands of mRNA targets, transporting mRNA from the nucleus to the cytoplasm and determining the fate of the transcript.

[0024] In recent years, numerous reports have confirmed that IGF2BP2 plays a pro-oncogene role in various malignancies, including hepatocellular carcinoma, esophageal adenocarcinoma, ovarian cancer, breast cancer, and glioblastoma. Huang et al. knocked down IGF2BPs in HeLa and HepG2 cells, resulting in inhibition of tumor cell proliferation, clonogenicity, and migration / invasion, similar to MYC silencing. Mu et al. reported that IGF2BP2 enhances the proliferation and invasion of glioma cells and accelerates their epithelial-mesenchymal transition. Studies in colon, ovarian, and breast cancer have shown that autoimmune responses targeting IGF2BP2 are closely associated with disease progression, and IGF2BP2 antibodies can be used as a marker for tumor screening, diagnosis, and prognosis. Studies have also found that IGF2BP2 can induce genomic instability in liver cancer cells, leading to increased tumor malignancy. However, the role and mechanism of IGF2BP2 in T-ALL remain unclear.

[0025] Based on this, first, the present invention provides the use of a reagent for detecting the expression of the IGF2BP2 gene in the preparation of a diagnostic reagent for acute T lymphocytic leukemia.

[0026] IGF2BP2 is involved in the development, progression, invasion, and metastasis of various solid tumors. The results of this study demonstrate that IGF2BP2 expression in bone marrow cells from T-ALL patients is significantly elevated compared to healthy controls. Downregulating IGF2BP2 expression in T-ALL cell lines significantly inhibits cell proliferation and induces apoptosis, while upregulating IGF2BP2 expression significantly promotes cell proliferation and reduces sensitivity to chemotherapeutic drugs. These results suggest that IGF2BP2 functions as an oncogene in acute T-lymphoblastic leukemia. Based on general research in this field, detecting IGF2BP2 expression is expected to be diagnostic for acute T-lymphoblastic leukemia.

[0027] In a specific embodiment, IGF2BP2 is significantly increased in bone marrow cells of T-ALL patients at diagnosis compared to healthy controls;

[0028] In a preferred embodiment, the diagnostic reagents include RT-PCR and qRT-PCR reagents for IGF2BP2 gene expression.

[0029] Secondly, the present invention provides the use of a substance that specifically targets and binds to IGF2BP2 in the preparation of a product for treating acute T lymphocytic leukemia.

[0030] To validate IGF2BP2 as a therapeutic target, the present invention conducted a computer-generated virtual screening targeting the previously reported KH34 domain of IGF2BP2 (the KH34 domain mediates the interaction between IGF2BP2 and RNA) to identify small molecule compounds that may interact with IGF2BP2. In vitro experiments were then used to further screen for small molecule inhibitors that could bind to and inhibit IGF2BP2 activity, confirming that they specifically bind to IGF2BP2 in T-ALL cells and inhibit T-ALL cell proliferation.

[0031] In a preferred embodiment, the substance that specifically targets and binds to IGF2BP2 is a substance that downregulates the expression of the IGF2BP2 gene, including but not limited to siRNA, shRNA, microRNA, etc.

[0032] Specifically, the treatment of acute T-lymphocytic leukemia includes one or more of inhibiting cell proliferation, preventing cells from dividing into S phase, and promoting cell apoptosis.

[0033] In a preferred embodiment, the substance that specifically targets and binds to IGF2BP2 is a small molecule compound that specifically targets and binds to IGF2BP2, such as JX5 and its derivatives.

[0034] The structural formula of JX5 is shown in Formula 1 below:

[0035]

[0036]

[0037] The structural formula of the JX5 derivative is shown in Formula II below:

[0038]

[0039] Preferably, R 1 ~R 28 Optional

[0040] OH / CX3 / CHX2 / CH2X / OCX3 / OCHX2 / OCH2X / CN / C(O)Y1 / C(O)OY 1 / C(O)NY 1 Y 2 / C(O)NHY 1 / NHC(O)Y 1 / NY 1 C(O)Y 2 / NY 1 C(O)OY 2 / NY 1 OY 2 / N3 / NY 1 C(O)Y 2 / NY 1 NY 2 Y 3 / SO4Y 1 / SO3Y 1 / SO2Y 1 / SO4NY 1 Y 2 / SO3NY 1 Y 2 / SO2NY 1 Y 2 / SF5;

[0041] Where X = H / Cl / F / Br / I;

[0042] Y 1 =Y 2 =Y 3 =OH / CCl / CBr3 / CF3 / CI3 / CH2Cl / CH2F / CH2Br / CH2I / CHCl2 / CHF2 / CHBr2 / CHI2 / CN / NH2 / COOH / CONH / NO2 / SH / SO3H / SO2HN2 / SO4H / HNNH2 / ONH2 / NHC (O)NHNH2 / NHC(O)NH2 / NSO2H / NHC(O)H / NHC(O)OH / NHOH / OCCI3 / OCBr3 / OCF3 / OCI3 / OCHCl2 / OCHBr2 / OCHF2 / OCHI2.

[0043] The third aspect of the present invention provides the use of JX5 and its derivatives as IGF2BP2 targeted inhibitors.

[0044] The role of IGF2BP2 in tumorigenesis and progression suggests it as a potential anti-tumor drug target, but currently no drugs targeting IGF2BP2 have been reported. IGF2BP2 contains two RNA recognition domains (RRM domains) and four hnRNP-K homology domains (KH domains). Studies have shown that the KH domains mediate the interaction between IGF2BP2 and target RNAs, while the RRM domains promote the stability of the IGF2BP2-RNA complex. Through database searches (Protein Data Bank), only a portion of the protein's spatial structure has been determined by crystal diffraction (KH34 domain, PDB: 6ROL).

[0045] JX5 and its derivatives, as targeted inhibitors of IGF2BP2, can not only be directly used to treat acute T-lymphocytic leukemia; the interaction (targeted binding) between JX5 and its derivatives and IGF2BP2 can be used for in vitro drug screening, construction of protein interaction models and other applications for non-disease diagnosis and treatment purposes.

[0046] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0047] Example 1 Analysis of the role of IGF2BP2 gene in T-ALL

[0048] IGF2BP2 plays an oncogenic role in various solid tumors and acute myeloid leukemia, but its role and mechanism in T-ALL remain unclear. Using T-ALL patient bone marrow cells and T-ALL cell lines, we confirmed that IGF2BP2 plays an oncogene role in T-ALL and identified it as a potential therapeutic target.

[0049] 1.1 IGF2BP2 expression in T-ALL patients

[0050] To clarify the expression of IGF2BP2 in T-ALL patients, this example collected 9 T-ALL patients and 6 healthy controls, and used qPCR to detect the expression of IGF2BP2 mRNA levels. The results showed that IGF2BP2 in the bone marrow cells of T-ALL patients was significantly increased compared with that in healthy controls ( Figure 1 ).

[0051] 1.2 Effects of downregulating IGF2BP2 expression on cell biological functions in T-ALL cell lines

[0052] In this example, siRNA was used to downregulate the expression of IGF2BP2 in two T-ALL cell lines, Jurkat and Molt4, and the effects on cell biological functions were verified by CCK8, cell cycle assays, and cell apoptosis assays. The results showed that downregulating the expression of IGF2BP2 in T-ALL cell lines could inhibit cell proliferation ( Figure 2 B), prevent cells from dividing into S phase ( Figure 2 C), promote cell apoptosis ( Figure 2 D).

[0053] Effect of IGF2BP2 on the pathogenesis of leukemia in T-ALL xenograft mouse models

[0054] In this example, the expression of IGF2BP2 was down-regulated in Jurkat cells. After tail vein injection into NSG mice, the leukemia load was observed. The results showed that IGF2BP2 could promote the onset of leukemia in T-ALL xenograft mouse models ( Figure 3 ).

[0055] Example 2 Drugs that specifically target and bind to IGF2BP2

[0056] Because elevated IGF2BP2 expression is highly correlated with the development and progression of T-ALL, novel targeted drugs targeting IGF2BP2 could be designed. We first conducted in silico screening using the KH34 domain of IGF2BP2 (which mediates its interaction with RNA) to identify small molecules that could interact with IGF2BP2. We then conducted in vitro experiments to identify small molecule inhibitors that could bind to and inhibit IGF2BP2 activity. These inhibitors were confirmed to bind to IGF2BP2 in T-ALL cells and inhibit T-ALL cell proliferation.

[0057] 2.1 In silico screening revealed that JX5 interacts with IGF2BP2

[0058] The present invention used the SPECS database (306,709 small molecule compounds, http: / / www.specs.net) for molecular docking-based virtual screening. The human IGF2BP2 protein structure was downloaded from the Protein Data Bank (http: / / www.rcsb.org / ) (PDB ID: 6ROL). Polar hydrogens and charges were added to the protein using AutodockTools 1.5.6, and the protein was converted to PDBQT format. The coordinates of the active pocket of the IGF2BP2 protein were set as follows: center_x = 31.471, center_y = -4.855, center_z = 11.12; size_x = 37.5, size_y = 41.25, size_z = 37.5. In addition, the parameter num_modes was set to 1. Unless otherwise specified, all other parameters were used at their default values. Finally, the SPECS database was split using Openbabel software and batch converted to PDBQT format using a script. Batch molecular docking was then performed using Autodock vina 1.1.2. According to the size of the binding energy, virtual evaluation and scoring are performed ( Figure 4 ).

[0059] 2.2 In vitro confirmation of the small molecule compound JX5 targeting IGF2BP2

[0060] The screening results from step 2.1 were evaluated based on their chemical structure diversity and binding energy. Twenty-two small molecules were selected for further confirmation of their interaction with IGF2BP2 using fluorescence titration. A 100 mM stock solution of the compound was prepared. 1.0 μL of the stock solution was added to 99 μL of buffer (the same buffer used for IGF2BP2 protein) and diluted to 1000 μM. 100 μL of a 5 μM IGF2BP2 protein solution was added to a 96-well plate, and an equal volume of buffer was added in parallel. Fixed volumes of compound solution (0, 3, and 6 μL for screening; 0, 1, 2, 4, 6, 8, 10, and 20 μL for confirming compound binding to IGF2BP2) were then added. The mixture was gently pipetted and incubated at 4°C for 10 minutes. Under 280nm wavelength excitation light, read the fluorescence value of each well (332nm), subtract the fluorescence value of the corresponding buffer well from the fluorescence value of the protein well (minus the optical interference of the compound), and observe whether the fluorescence intensity decreases with the increase of the compound amount and conforms to the dose-effect relationship. The fluorescence titration results show that JX2, JX5, JX14 and JX19 can bind to IGF2BP2 ( Figure 4 ).

[0061] 2.3 Effects of compound JX5 on the biological functions of T-ALL cells

[0062] Based on the fluorescence titration results, this example further evaluated the effects of four small molecule compounds on cell proliferation in HPB-ALL (low expression of IGF2BP2) and Jurkat (high expression of IGF2BP2) cell lines, and found that JX5 specifically inhibited the proliferation of Jurkat cells, suggesting that JX5 targeted and bound to intracellular IGF2BP2 and inhibited its activity. On this basis, this example analyzed the effect of JX5 on cell proliferation in IGF2BP2 overexpression or knockout cells and respective control cells, and verified the effect of JX5 on the IGF2BP2 mRNA level in cells, confirming at the cellular level that JX5 targeted and inhibited IGF2BP2 ( Figure 5 , 6, 7, 8).

[0063] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Application of reagents for detecting IGF2BP2 gene expression in the preparation of diagnostic reagents for acute T-lymphocytic leukemia; IGF2BP2 was significantly increased in bone marrow cells of patients with acute T-lymphoblastic leukemia at diagnosis compared with healthy controls.

2. The use according to claim 1, characterized in that The diagnostic reagent includes an RT-PCR reagent for the expression of the IGF2BP2 gene.

3. The use according to claim 1, characterized in that The diagnostic reagent includes a qRT-PCR reagent for the expression of the IGF2BP2 gene.

4. Application of substances that specifically target and bind to IGF2BP2 in the preparation of products for the treatment of acute T-lymphocytic leukemia; The substance that specifically targets and binds to IGF2BP2 is a small molecule compound that specifically targets and binds to IGF2BP2; The small molecule compound is JX5, and the structural formula of JX5 is shown in Formula 1: Formula I.

5. The use according to claim 4, characterized in that The treatment of acute T-lymphocytic leukemia includes one or more of inhibiting cell proliferation, preventing cells from dividing into S phase, and promoting cell apoptosis.

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