Anti-leukemia small-molecule compound TIP-20 targeting FLT3 and application of anti-leukemia small-molecule compound TIP-20

Abstract

The invention provides an anti-leukemia small molecule compound TIP-20 targeting FLT3 and application of the anti-leukemia small molecule compound TIP-20, and belongs to the technical field of biological medicine. The invention develops a novel FLT3 kinase inhibitor TIP-20, which can enhance the antigen presentation effect of FLT3-ITD leukemia cells, can be combined with FLT3 protein, stabilizes the structure of FLT3, and further inhibits FLT3 phosphorylation and downstream pathway activation. The TIP-20 has an obvious inhibiting effect on the growth of leukemia cell strains, and can inhibit the growth of leukemia cells with FLT3 mutation by inhibiting phosphorylation of FLT3, promoting cell apoptosis and cycle arrest. Moreover, FLT3 wild type cells have good tolerance to TIP-20, have a wide therapeutic window with FLT3-ITD mutated acute myelogenous leukemia cells, and are expected to bring a better choice for treatment of FLT3 mutated AML patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a small molecule compound TIP-20 targeting FLT3 for the treatment of leukemia and its applications. Background Technology

[0002] Acute myeloid leukemia (AML) is a major type of leukemia, accounting for approximately 80% of adult acute leukemia cases. Over the past 40 years of treatment, the progression of different AML subtypes has varied significantly. Acute promyelocytic leukemia (AML) has seen a significant increase in cure rates due to the introduction of targeted therapy. However, progress in treating other AML subtypes has been quite slow, with treatment still primarily relying on high-dose cytarabine chemotherapy or allogeneic hematopoietic stem cell transplantation, combined with consolidation therapy based on risk stratification. Under this traditional treatment strategy, the 5-year survival rate for AML patients under 60 years of age is approximately 40%, while the 5-year survival rate for patients over 60 years of age is less than 10%. Therefore, actively exploring targeted therapies is a crucial direction for improving the prognosis of this type of AML.

[0003] FLT3 (Fms-like tyrosine kinase 3), a member of the receptor tyrosine kinase class III (RTK III) subfamily, has a mutation rate as high as 30% in adult AML. It mainly exists in two types of mutations: internal tandem repeat (ITD) mutations, accounting for approximately 2 / 3, and kinase domain (TKD) point mutations, accounting for approximately 1 / 3. Both types of mutations are gain-of-function mutations—allowing the FLT3 receptor to continuously autophosphorylate without ligand dependence, abnormally activating signaling pathways, and thus participating in the development and progression of leukemia and maintaining the abnormal proliferative capacity of tumor cells. Meanwhile, FLT3-ITD-positive AML patients, compared to mutation-negative patients, have a worse response to induction chemotherapy, a higher relapse rate, and significantly shorter relapse-free survival and overall survival. Given that FLT3 is the most common molecular mutation in AML and that conventional treatments for mutated patients have a poor prognosis, it has become the most attractive therapeutic target in the field of AML and a core direction for improving the prognosis of these patients.

[0004] Currently, more than ten small molecule inhibitors targeting the FLT3 receptor kinase have entered clinical trials or been approved for marketing. Although patients with FLT3-mutant AML have a high response rate to these targeted inhibitors, the off-target side effects remain a significant and challenging issue in clinical application, greatly limiting treatment benefits. Small molecule compound libraries have always been an important source for drug research and development. Multiple studies have shown that small molecule compounds possess a wide range of pharmacological effects and various biological activities, such as anti-inflammatory, antioxidant, and tumor cell apoptosis-promoting effects. Therefore, developing specific FLT3 inhibitors based on the ATP-binding site of the inactive FLT3 protein not only has significant clinical value but also broad application prospects, potentially providing a better treatment option for patients with FLT3-ITD AML. Summary of the Invention

[0005] The purpose of this invention is to provide a small molecule compound TIP-20 targeting FLT3 for leukemia and its application, which can inhibit the growth of leukemia cells with FLT3 mutations by inhibiting FLT3 phosphorylation, promoting apoptosis and cell cycle arrest.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a small molecule compound, TIP-20, targeting FLT3 to treat leukemia. The molecular formula of TIP-20 is C2. 18 H 17 N7, CAS: 2306751-39-1, chemical structural formula is shown in formula (1): Equation (1).

[0007] The present invention also provides the application of the above-mentioned small molecule compound TIP-20 in the preparation of a drug for treating acute myeloid leukemia.

[0008] The beneficial effects of this invention compared to the prior art are as follows: (1) The present invention developed a novel FLT3 kinase inhibitor TIP-20. TIP-20 can enhance the antigen presentation of FLT3-ITD leukemia cells, bind to FLT3 protein, stabilize the structure of FLT3, enhance the thermal stability of FLT3 protein, and further inhibit FLT3 phosphorylation and downstream pathway activation.

[0009] (2) The FLT3 kinase inhibitor TIP-20 of the present invention has a significant inhibitory effect on the growth of leukemia cell lines. It can inhibit the growth of leukemia cells with FLT3 mutations by inhibiting FLT3 phosphorylation, promoting apoptosis and cell cycle arrest. In addition, FLT3 wild-type cells have good tolerance to TIP-20 and have a broad therapeutic window with FLT3-ITD-mutant acute myeloid leukemia cells, which is expected to bring better treatment options for patients with FLT3-mutant AML. Attached Figure Description

[0010] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0011] Figure 1 This is a graph showing the cell viability inhibition of TIP-20 on 32D cells and 32D-ITD stable transgenic cells in Experiment Example 1 of this invention, where IC50... 50 Represents the half-inhibitory concentration; Figure 2 The graph shows the inhibitory effect of TIP-20 on the proliferation of leukemia cell lines with or without FLT3 mutation in Experiment Example 1 of this invention. In the graph, A represents quinzatinib (AC220), B represents TIP-20, and *** represents p<0.001. Figure 3 This is a three-dimensional schematic diagram of the binding mode between TIP-20 and FLT3 protein in Experimental Example 1 of the present invention; Figure 4 The inhibitory activity of TIP-20 against FLT3 kinase in Experimental Example 1 of this invention; Figure 5 This is an example of the effect of TIP-20 on the thermostability of FLT3 protein in the 293T stable cell line in Experiment 1 of this invention. A is the FLT3 detection result, and B is a line graph of the detection result. Figure 6 The inhibitory effect of different concentrations of AC220 (A) and TIP-20 (B) on phosphorylation level in Experimental Example 1 of this invention; Figure 7 The apoptosis-promoting effect of TIP-20 on 32D-ITD, MV4-11 and Molm13 cells in Experimental Example 1 of this invention is shown in Figure A, where A is a representative flow cytometry atlas and B is the statistical analysis. Figure 8The cell cycle arrest effect of TIP-20 on 32D-ITD, MV4-11 and Molm13 cells in Experiment Example 1 of this invention is shown in Figure A, where A is a representative flow cytometry atlas and B is a statistical analysis. Figure 9 These are bone marrow smears from mice in different treatment groups in Experiment Example 2 of this invention; Figure 10 The spleen size and weight of mice in different treatment groups in Experiment Example 2 of this invention; Figure 11 The proportion of hCD45+ cells in the bone marrow and spleen of mice in different treatment groups in Experiment Example 2 of this invention; Figure 12 The concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (UREA), and creatinine (CREA) in the serum of mice in different treatment groups in Experiment Example 2 of this invention. Detailed Implementation

[0012] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0013] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0014] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0015] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0016] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0017] Example 1 Example 1 of this invention prepared a stable 32D-FLT3-ITD cell line. The specific steps are as follows: (1) Material preparation Experimental cells: Human Molm-13, MV4-11, THP-1, K562, HL60, Kasumi-1, 293T cells and mouse 32D cells were all purchased from Beina Biotechnology Co., Ltd.

[0018] Experimental reagents: AC220 and TIP-20 (CAS: 2306751-39-1) were purchased from Shanghai Taosu Biochemical Technology Co., Ltd.; Recombinant Murine IL-3 was purchased from PEPROTECH (USA); CCK8 reagent was purchased from Tongren Chemical Co., Ltd. (Japan); Annexin V-APC apoptosis detection kit was purchased from biogems (USA); Anti-pFLT3, p-STAT5, and STAT5 antibodies were purchased from Cell Signaling Technology (USA); Horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) was purchased from Beyotime (China); pLenti-III-EF1α (LV043) plasmid vector was purchased from Applied Biological Materials (abm) Inc. (Canada). (2) Construction of FLT3-ITD mutant plasmid The pLenti-III-EF1α(LV043) plasmid vector was purchased from Gemma Biosciences and used as a template for PCR amplification. The FLT3 fragment was prepared using HiFi PCR premixed reagents for cloning amplification. Subsequently, these PCR fragments were integrated into the LV043 vector using the In-Fusion HD Cloning Plus kit to obtain the FLT3-ITD mutant plasmid.

[0019] (3) FLT3-ITD lentivirus packaging 293T cells were seeded in 10cm cell culture dishes and passaged until they reached the logarithmic growth phase. After adjusting the cell line condition to be optimal, the cells were allowed to grow uniformly until they occupied 70%–80% of the dish bottom volume (cell count 5–7 × 10⁶). 6 (pieces / dish).

[0020] Two hours before the start of virus packaging, discard the original culture medium and replace it with 5 mL of fresh DMEM complete culture medium. Then, package the lentivirus according to the system shown below: Take two 1.5 mL EP tubes, label tubes A and B respectively, and add 500 μL of serum-free DMEM culture medium to each. First, add 40 μL of liposome Lipo 2000 to tube B and gently pipette to mix. Take tube A and add the packaging system shown in Table 1, and gently pipette to mix.

[0021] Table 1 Lentiviral Packaging Systems

[0022] Slowly add the liquid from tube B dropwise to tube A until all liquid is added. Gently rinse and mix three times, then incubate at 37°C for 30 minutes. Gently add the mixture from tubes A and B dropwise to a cell culture dish, mixing thoroughly using the cross-hatching method. Incubate for 8 hours. After 8 hours of culture, discard all the original culture medium into a waste liquid processor containing sterilization solution, and add 5 mL of fresh DMEM complete culture medium to complete the cell culture medium replacement.

[0023] After 24 hours of culture, the first collection of lentivirus-containing culture media was performed in 15 mL centrifuge tubes, sealed, and stored at 4°C, with 5 mL of fresh DMEM complete medium added. The virus was collected a second and third time after 48 and 72 hours of culture, respectively. All collected virus solutions were centrifuged at 1500 rpm at 4°C for 15 minutes. The supernatant was filtered through a 0.22 μm filter to remove cell debris and other impurities, yielding packaged FLT3-ITD mutant lentivirus. This lentivirus was aliquoted and stored at -80°C for later use.

[0024] (4) FLT3-ITD lentivirus infection of mouse 32D cells and screening of stable transgenic strains The FLT3-ITD mutant lentivirus packaged in step (3) was used to infect mouse 32D cells. After 72 hours of infection, the cells were cultured in a medium containing puromycin until all negative control (uninfected cells) cells died.

[0025] Well-grown 32D cells were lysed, and Western blotting was used to detect HA-tag expression to identify whether the cells stably expressed FLT3 mutant protein. Cells that stably expressed FLT3 wild-type / mutant protein were identified as mouse 32D-FLT3-ITD stable cell line.

[0026] Experimental Example 1 Experimental Example 1 of this invention uses TIP-20 to study the inhibitory effect of TIP-20 on FLT3. The specific steps are as follows: (1) Drug screening Based on the co-crystallization structure of FLT3 (PDB: 4xuf), using the ATP-binding domain of FLT3 as the binding target region, 159 lead small molecule compounds were obtained through high-throughput screening in the ChemDivCN library. After multiple screenings at the cellular level and biochemically, TIP-20 (C...) was selected. 18 H 17 N7; CAS 2306751-39-1).

[0027] (2) Cell viability detection 32D cells were used as the control group, and the 32D-ITD stable transgenic cell line prepared in Example 2 was used as the experimental group. Figure 2 Different concentrations of TIP-20 solutions were prepared (100 μM was the highest concentration, serially diluted 2-fold, with 9 concentration gradients, and incubated for 72 hours). CCK8 susceptibility testing was performed to detect cell viability in both groups at different TIP-20 concentrations. Results are as follows: Figure 1 As shown.

[0028] Figure 1 The results showed that TIP-20 had a significantly better inhibitory effect on stable 32D-ITD mutant cells than on normal cells without FLT3 mutations, and it showed a more significant inhibitory effect on 32D-ITD mutant cells at multiple concentration gradients.

[0029] (3) Inhibition effect detection Using quezartinib (AC220) as a positive control, two groups were established: non-FLT3 mutant leukemia cell groups (THP-1, HL60, K562, and Kasumi-1) and FLT3 mutant leukemia cell groups (MV-4-11 and Molm13). Figure 2 Different concentrations of TIP-20 solutions and AC220 solutions were prepared and subjected to CCK8 drug sensitivity testing to detect the cytotoxic specificity of TIP-20. The results are as follows: Figure 3 As shown.

[0030] Figure 2 The results showed that, in FLT3-mutant leukemia cell lines (MV-4-11 and Molm13) and non-FLT3-mutant leukemia cell lines (THP-1, HL60, K562, and Kasumi-1), the half-maximal inhibitory concentration (IC50) of TIP-20 for FLT3-mutant myeloid leukemia cell lines (MV-4-11 and Molm13) was significantly lower than that for non-FLT3-mutant myeloid leukemia cell lines (THP-1, HL60, and K562). Figure 2 Overall, TIP-20 significantly inhibited the growth of leukemia cell lines with FLT3-ITD mutations.

[0031] (4) Specific binding of TIP-20 to FLT3 protein Virtual molecular docking of TIP-20 with the FLT3 protein was performed using Schrödinger Maestro, and the results are as follows: Figure 3 As shown. FLT3 kinase inhibition was quantified by homogeneous time-resolved fluorescence (HTRF) assay, which monitors the phosphorylation of biotin-labeled tyrosine kinase substrate peptides. Assay conditions (substrate, ATP and enzyme levels, and reaction time) were optimized beforehand. The test compound was prepared as a DMSO stock solution and diluted to reaction buffer (50 mM HEPES, pH 7.0; 0.1 mM sodium orthovanadate; 0.01% BSA; 0.02% NaN3; 5 mM MgCl2; 5 mM MnCl2; 1 mM DTT). 4 μL of the compound (specified concentration) was added to each well of a white 96-well plate, followed by 2 μL of FLT3 kinase (kinase buffer, 1 ng / μL). After a 10-minute pre-incubation, the reaction was initiated by adding 2 μL of substrate (final concentration 1 μM) and 2 μL of ATP (final concentration 100 μM). After incubating the mixture at 37°C for 60 minutes, the mixture was analyzed by adding 10 μL of the test solution (composed of 5 μL Eu). 3+ - A mixture of cryptokinin-labeled anti-TK antibody and 5 μL streptavidin-XL665 (final concentration 125 nM) was quenched to detect phosphorylated peptides. After incubation at 37°C for 1 hour, time-resolved FRET signals (excitation wavelength 337 nm and dual emission wavelengths) were recorded using a BioTek Cytation 3 multimode reader. The results are as follows: Figure 4 As shown.

[0032] Figure 3 The results show that TIP-20 can specifically bind to the FLT3 protein (forming hydrogen bonds with FLT3 protein Cys694 and Glu661). This binding can inhibit the activation of FLT3 kinase by competitively occupying the ATP-binding pocket of the FLT3 protein. Figure 4 ).

[0033] Further intracellular thermal displacement experiments were conducted using HEK-293T cells (1×10⁻⁶) stably expressing FLT3-ITD. 8Cells were treated overnight with TIP-20 (10 μM, 20 μM, 50 μM) or DMSO. Cell lysates were lysed with RIPA buffer, aliquoted, and heated for 3 minutes in a temperature gradient of 40–58.4 °C. Protein detection was performed using Western blotting. For isothermal dose-response analysis, lysates were prepared after treatment with TIP-20 (0–100 μM) at 51 °C. Band intensities were quantitatively analyzed using ImageJ software, and statistical analysis was performed using GraphPad Prism 8.0 software. Results are shown below. Figure 5 As shown.

[0034] Figure 5 The results show that TIP-20 can stabilize the structure of FLT3 and enhance its thermal stability, which indirectly demonstrates the binding of TIP-20 to the FLT3 protein.

[0035] (5) TIP-20 can effectively reduce the phosphorylation level of FLT3. FLT3, a member of the tyrosine kinase family, promotes cell proliferation and survival by phosphorylating downstream signaling proteins through its kinase activity. The phosphorylation capacity of FLT3 partially reflects its activation ability of downstream signaling pathways. Using quezartinib (AC220) as a positive control, the effects of different concentrations of TIP-20 on the downstream pathways of FLT3 were investigated. 32D-ITD stable cell lines were treated with TIP-20 and AC220 (1 μM, 2 μM, and 5 μM) for 6 h, and cells were harvested. After protein lysis, Western blotting was used to detect FLT3 and its downstream STAT5 and their phosphorylation levels. GADPH was used as a loading control. The results are as follows: Figure 6 As shown.

[0036] Figure 6 The results showed that TIP-20 could significantly inhibit FLT3 and downstream STAT5 phosphorylation. (6) TIP-20 has a significant pro-apoptotic and cell cycle arrest effect on FLT3 mutant cells. Flow cytometry was used to detect the apoptosis-inducing and cell cycle arrest effects of TIP-20 on 32D-ITD stable cell lines, MV4-11, and Molm13. The results are as follows: Figure 7 , 8 As shown.

[0037] Figure 7 The results showed that TIP-20 could induce apoptosis in 32D-ITD stable cell line, MV4-11, and Molm13 cells to a certain extent, with a significant killing effect. Cell cycle analysis indicated that TIP-20 mainly caused G0 / G1 phase arrest in 32D-ITD cells. Figure 8 ).

[0038] Experimental Example 2 Test Example 2 of this invention tested the in vivo activity and drug toxicity of TIP-20, and the specific steps are as follows: A CDX tumor model was established by injecting the FLT3 mutant AML cell line MV4-11 into 15 NCG immunodeficient mice via tail vein injection (2×10⁶ cells / year). 6 In a study conducted on mice (cells / mouse), 15 mice were randomly divided into three groups 14 days after tail vein injection. The mice were administered TIP-20 (30 mg / kg or 100 mg / kg, 200 μL, once daily) or 20% cyclodextrin (200 μL, once daily) via gavage, respectively. After 14 days of treatment, all mice were sacrificed. Spleens were harvested for size observation, bone marrow (BM) samples were collected to detect the proportion of hCD45+ cells, and serum was collected for liver and kidney function testing. Results are as follows: Figures 9-12 As shown.

[0039] The results showed that TIP-20 could reduce the proportion of blast cells in the bone marrow. Figure 9 ), improves splenomegaly in tumor-bearing mice ( Figure 10 ), reduce tumor burden in bone marrow ( Figure 11 Furthermore, continuous drug use did not increase the concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (UREA), and creatinine (CREA) in mouse serum. Figure 12 This indicates that TIP-20 has no significant liver or kidney toxicity.

[0040] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An anti-leukemic small molecule compound targeting FLT3, TIP-20, characterized in that, The molecular formula of TIP-20 is C 18 H 17 N7, CAS: 2306751-39-1, chemical structural formula is shown in formula (1): Formula (1).

2. The use of the small molecule compound TIP-20 of claim 1 in the preparation of a medicament for treating acute myeloid leukemia.

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