Use of flavonoid small molecule compounds in the preparation of drugs for regulating WDR1 and / or treating leukemia

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a flavone small molecule compound in preparation of a drug for regulating WDR1 and / or treating leukemia. The flavone small molecule compound comprises Gambogic amide, Morellic acid, Flavopiridol hydrochloride, Gambogic Acid and / or Methylophiopogonanone B. The application research finds that the flavone small molecule compound such as Gambogic amide can effectively inhibit the activity of acute myeloid leukemia stem cells, and does not damage normal hematopoietic stem cells. The flavone small molecule compound is combined with WDR1 protein specifically expressed in the leukemia stem cells, induces cytoskeleton depolymerization, and further promotes the occurrence of leukemia stem cell apoptosis. The application provides a new direction for prevention and treatment of AML.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a flavonoid small molecule compound in the preparation of drugs that regulate WDR1 and / or treat leukemia. Background Technology

[0002] Acute myeloid leukemia (AML) is a blood disorder characterized by the arrest of myeloid progenitor cell differentiation, leading to abnormal proliferation of cells at the progenitor stage. Although current chemotherapy regimens can achieve a cure rate of up to 70% for AML patients, the presence of AML LSCs derived from AML patients still leads to relapse. Furthermore, AML LSCs and normal hematopoietic stem cells (HSCs) share certain similarities, such as low cell number, quiescent state, self-renewal potential, and similar cell surface marker molecules. This results in existing chemotherapy drugs causing toxic damage to normal HSCs while simultaneously eliminating AML LSCs. Therefore, exploring ways to efficiently and specifically eliminate AML LSCs to significantly improve the cure rate for AML patients remains a challenging and hotly debated research topic.

[0003] When developing drugs to target and clear AML LSCs, it is crucial to fully consider the unique characteristics of AML LSCs to achieve the desired results. Existing research has revealed that AML LSCs, compared to normal HSCs, exhibit specific characteristics such as cell adhesion and oxidative phosphorylation. Therefore, intervening in the cytoskeleton and metabolic pathways of AML LSCs may be effective in clearing AML LSCs without damaging normal HSCs. For example, based on the fact that AML LSCs are more dependent on oxidative phosphorylation than normal HSCs, small molecules that inhibit mitochondria or Veneclare targeting electron transport chain activity and the tricarboxylic acid cycle have been developed. However, GDYO, which targets the AML cytoskeleton, binds to ITGB2 and MRC2 and is absorbed by AML cells, inhibiting F-ACTIN polymerization and inducing apoptosis. However, the effectiveness of GDYO in AML LSCs is unclear, and research on small molecule compounds related to cytoskeleton remodeling or metabolic pathways in AML LSCs is also incomplete. Therefore, exploring small molecule compounds that specifically clear AML LSCs without damaging normal HSCs is urgently needed.

[0004] Flavonoids are bioactive compounds extracted from plants. They and their derivatives participate in apoptosis, cell metabolism, cytoskeleton regulation, and epigenetic regulation, thus playing an important role in the treatment of diseases such as inflammation, aging, Alzheimer's disease, and cancer. Patent CN108354932A reported the application of the flavonoid GL-V9 in the preparation of an anti-leukemia drug, finding that GL-V9 induces the differentiation of acute myeloid leukemia (AML) cells into dendritic cells. However, whether flavonoid natural products have the effect of clearing AML LSCs is currently unclear, and the efficacy of different types of flavonoids varies significantly.

[0005] Therefore, it is necessary to conduct in-depth research on the application of flavonoid small molecule compounds in clearing AML LSCs, in order to discover small molecule compounds that can efficiently and specifically clear AML LSCs, thereby improving the cure rate of AML patients or reducing the recurrence rate of AML patients. Summary of the Invention

[0006] In view of this, one object of the present invention is to provide the use of flavonoid small molecule compounds or their solvates, optical isomers, polymorphs or pharmaceutically acceptable salts in the preparation of drugs that regulate WDR1. These flavonoid small molecule compounds regulate WDR1 by interacting with the WDR1 protein.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The use of flavonoid small molecule compounds or their solvates, optical isomers, polymorphs or pharmaceutically acceptable salts in the preparation of drugs regulating WDR1, wherein the flavonoid small molecule compounds include one or more of Gambogic amide, Morellitic acid, Flavopiridol hydrochloride, Gambogic Acid, and Methylophiopogonanone B; the structural formulas of each flavonoid small molecule compound are as follows:

[0009]

[0010] .

[0011] Preferably, the flavonoid small molecule compound is Gambogic amide.

[0012] Preferably, the regulation includes positive regulation and / or negative regulation, with negative regulation being the preferred method.

[0013] A second objective of this invention is to provide the use of flavonoid small molecule compounds or their solvates, optical isomers, polymorphs or pharmaceutically acceptable salts in the preparation of medicaments for the treatment of leukemia.

[0014] To achieve the above objectives, the present invention adopts the following technical solution:

[0015] The use of flavonoid small molecule compounds or their solvates, optical isomers, polymorphs or pharmaceutically acceptable salts in the preparation of medicaments for the treatment of leukemia, wherein the flavonoid small molecule compounds include any one or more of Gambogicamide, Morellitic acid, Flavopiridol hydrochloride, Gambogic Acid, and Methylophiopogonanone B.

[0016] Preferably, the treatment comprises administration of a flavonoid compound at a dose of 0.2 μM to 0.8 μM; wherein, before treatment, the subject or experimental animal has a baseline level of leukemia stem cell viability of more than 95%, a stem cell proportion of more than 8%, a late apoptosis baseline level of less than 25%, a cell differentiation baseline level of less than 40%, and a survival time of ≤48 days; after treatment with the flavonoid small molecule compound, the subject or experimental animal has a stem cell viability of less than 60%, a stem cell proportion of less than 2%, a late apoptosis level of more than 55%, a cell differentiation baseline level of more than 60%, and a survival time of more than 48 days.

[0017] Preferably, the treatment comprises administration of a dose of 0.4 μM to 0.8 μM flavonoids.

[0018] Preferably, the treatment comprises administration of a dose of 0.6 μM to 0.8 μM flavonoids.

[0019] Preferably, the subjects or experimental animals have 100% leukemia stem cell viability, a stem cell ratio of 10%, a cell differentiation level of 40%, a late apoptosis level of 20%, and an AML mouse survival time of 48 days before treatment.

[0020] Preferably, after treatment with the flavonoid small molecule compound, the leukemia stem cell viability of the subject or experimental animal is 0%, the stem cell ratio is 0%, the cell differentiation level is 70%, the late apoptosis level is 60%, and the survival time of AML mice is greater than 48 days.

[0021] Preferably, the leukemia includes acute myeloid leukemia and relapsed / refractory acute myeloid leukemia.

[0022] Preferably, the acute myeloid leukemia is caused by WDR1-specific high expression.

[0023] Preferably, the flavonoid small molecule compound is Gambogic amide.

[0024] Preferably, the use of the flavonoid compound or its solvates, optical isomers, polymorphs or pharmaceutically acceptable salts thereof in the preparation of medicaments for delaying the progression of leukemia, clearing AML LSCs, inhibiting the cellular activity of AML LSCs and / or inducing apoptosis of AML LSCs.

[0025] This invention has found that flavonoid small molecule compounds such as Gambogic amide can effectively inhibit the activity of AML LSCs, reduce the proportion of AML LSCs, decrease membrane potential, and regulate cell apoptosis through interaction with WDR1, which is of great significance for the treatment of relapsed and refractory acute myeloid leukemia.

[0026] Preferably, the AML LSCs include CD34 + AML cells.

[0027] Preferably, the CD34 + AML cells include KG-1a and / or KASUMI-1.

[0028] A third objective of this invention is to provide the use of flavonoid small molecule compounds or their solvates, optical isomers, polymorphs or pharmaceutically acceptable salts in the preparation of medicaments for treating diseases caused by WDR1.

[0029] To achieve the above objectives, the present invention adopts the following technical solution:

[0030] The use of flavonoid small molecule compounds or their solvates, optical isomers, polymorphs or pharmaceutically acceptable salts in the preparation of medicaments for treating diseases caused by WDR1, wherein the flavonoid small molecule compounds include one or more of Gambogicamide, Morellitic acid, Flavopiridol hydrochloride, Gambogic Acid, and Methylophiopogonanone B.

[0031] Preferably, the diseases caused by WDR1 include acute myeloid leukemia and refractory relapsed leukemia.

[0032] Preferably, the flavonoid small molecule compound is Gambogic amide.

[0033] The fourth objective of this invention is to provide a pharmaceutical composition for the prevention or treatment of diseases caused by WDR1.

[0034] To achieve the above objectives, the present invention adopts the following technical solution:

[0035] A pharmaceutical composition for the prevention or treatment of diseases caused by WDR1, the pharmaceutical composition comprising a therapeutically effective amount of a small molecule flavonoid compound or its solvate, optical isomer, polymorph or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; the small molecule flavonoid compound comprising one or more of Gambogic amide, Morellitic acid, Flavopiridol hydrochloride, Gambogic Acid, and Methylophiopogonanone B.

[0036] Preferably, the flavonoid small molecule compound is Gambogic amide.

[0037] Preferably, the excipients include one or more of the following: diluents, fillers, binders, wetting agents, absorption promoters, surfactants, lubricants, stabilizers, flavoring agents, sweeteners, and colorings.

[0038] Preferably, the dosage form of the pharmaceutical composition includes one or more of the following: liquid formulation, solid formulation, semi-solid formulation, and gaseous formulation.

[0039] The beneficial effects of this invention are as follows:

[0040] 1. This invention has found that five flavonoid compounds—Gambogic amide, Morellitic acid, Flavopiridol hydrochloride, Gambogic Acid, and Methylophiopogonanone B—all have inhibitory effects on AML cell activity. Among them, Gambogic amide has an inhibitory effect on CD34. + The inhibitory effect on AML cell activity was more significant than that of cytarabine.

[0041] 2. This invention has discovered that the flavonoid small molecule compound Gambogic amide is suitable for the specific elimination of leukemia stem cells from humans and mice, exhibiting a highly efficient ability to induce apoptosis in leukemia stem cells both in vivo and in vitro. Experimental results show that Gambogic amide, within a dosage range of 0.28 μM to 0.6 μM, can effectively inhibit the activity of AML LSCs and maintain its efficacy for up to six days. Furthermore, Gambogic amide showed a significant effect in inhibiting the proportion of AML LSCs and delaying the progression of AML in mouse models.

[0042] 3. This invention utilizes bioinformatics analysis and cell heat transfer experiments to further demonstrate that Gambogic amide can remodel the cytoskeleton by binding to WDR1, a cytoskeleton regulatory protein specifically highly expressed in AML LSCs, ultimately leading to a decrease in AML LSC activity and delaying the onset of AML. This technology has significant implications for the treatment of relapsed / refractory AML patients.

[0043] 4. Existing technologies cannot completely eliminate heterogeneous leukemia stem cells from various sources and have significant toxic side effects on the normal hematopoietic system. This invention has found that Gambogic amide, while eliminating leukemia stem cells, has minimal impact on normal hematopoietic stem cells and very low toxic side effects, demonstrating significant clinical translational value. Attached Figure Description

[0044] Figure 1 This is a graph showing the screening process data for flavonoid small molecule chemicals, where a represents the treatment of CD34 with 518 flavonoid compounds. + Cell viability score of AML cells after treatment; b represents CD34 cells treated with five candidate flavonoids. + Cell viability analysis of AML cells (KG-1a); c represents CD34 cells treated with five candidate flavonoids. + Cell viability analysis diagram of AML cells (KASUMI-1).

[0045] Figure 2 Gambogic amide, a small molecule flavonoid, is present in human CD34. + The figure shows the results of the action analysis in AML cells, where a represents CD34 treated with Gambogic amide. + IC50 analysis of leukemia stem cells (KG-1a); b represents CD34 after short-term treatment with Gambogic amide. + Cell viability analysis of leukemia stem cells (KG-1a); c represents CD34 cells treated with Gambogicamide for a short time. + Cell viability analysis of leukemia stem cells (KASUMI-1); d represents CD34 cells treated with Gambogic amide for an extended period. + Cell viability analysis of leukemia stem cells (KG-1a); e represents CD34 cells treated with Gambogic amide for an extended period. + Cell viability analysis of leukemia stem cells (KASUMI-1) after transplantation; f represents CD34 cells treated with Gambogic amide in vivo. + Figure 1. Survival analysis of AML mice after leukemia stem cell (KG-1a) treatment.

[0046] Figure 3 Figure 1 shows the results of the analysis of the role of the flavonoid small molecule compound Gambogic amide in mouse AML LSCs. Figure 2 shows the flow cytometry analysis of the apoptosis level of AML LSCs in vivo after Gambogic amide treatment in MLL-AF9-induced AML mice; Figure 3 shows the flow cytometry analysis of the change in the proportion of AML LSCs in vivo after Gambogic amide treatment in MLL-AF9-induced AML mice; Figure 4 shows the flow cytometry analysis of the maturation and differentiation level of AML LSCs in vivo after Gambogic amide treatment in MLL-AF9-induced AML mice; and Figure 5 shows the survival analysis of AML mice after Gambogic amide treatment in MLL-AF9-induced AML mice.

[0047] Figure 4 The figures show the results of the toxicity analysis of the flavonoid small molecule compound Gambogic amide on normal hematopoietic stem cells. Figure a shows the cell viability analysis of peripheral blood cells from healthy individuals after treatment with five candidate flavonoid compounds; figure b shows the proportion of hematopoietic stem cells after treatment with peripheral blood cells from healthy individuals; figure c shows the proportion of bone marrow hematopoietic stem cells after treatment with Gambogic amide compound from healthy mice; figure d shows the proportion of bone marrow cells after treatment with Gambogic amide compound from healthy mice; figure e shows the proportion of myeloid B cells, T cells, and nuclei of myeloid cells in bone marrow after treatment with Gambogic amide compound from healthy mice; and figure f shows the proportion of erythroid cells in bone marrow after treatment with Gambogic amide compound from healthy mice.

[0048] Figure 5 This diagram shows the results of an analysis of the apoptosis induced by the flavonoid small molecule compound Gambogic amide through partial binding to the specific protein WDR1 in AML LSCs. Figure a shows a bar chart analyzing the mRNA expression level of WDR1, the target gene of Gambogic amide, in normal hematopoietic stem-progenitor cells and leukemia stem-progenitor cells; figure b shows a Western blot diagram of cell heat transfer analyzing the affinity between Gambogic amide and the target protein WDR1; figure c shows a flow cytometry diagram of mitochondrial membrane potential after treatment with F-action assembly agent (Gambogic amide) or deassembly agent (Jas); figure d shows a bar chart of the analysis results in figure c; and figure e shows a fluorescence staining diagram of Caspase3 apoptosis levels in CD34+ AML LSCs cells treated with Gambogic amide.

[0049] Figure 6 A comparison of the effects of Gambogic amide and the existing chemotherapy drug cytarabine on inhibiting AML LSCs.

[0050] Figure 7 This is a schematic diagram of Gambogic amide targeting and eliminating leukemia stem cells (AML LSCs). Gambogic amide can specifically target and bind to the WDR1 protein, which is specifically highly expressed in AML LSCs, remodeling the cytoskeleton, inducing an increase in the level of the apoptosis protein Caspase3, and promoting apoptosis. At the same time, since normal hematopoietic stem cells (HSCs) express WDR1 at low levels, Gambogic amide cannot induce apoptosis in normal cells by binding to WDR1. Detailed Implementation

[0051] The technical solution of the present invention will be described more clearly and completely below with reference to specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the protection scope of the present invention.

[0052] To enhance understanding of the present invention, certain key technical and scientific terms will be clearly defined below. Unless otherwise specified herein, all other technical and scientific terms shall follow their generally accepted and understood meanings within the art to which this invention pertains. It should be noted that the terminology used herein is intended to describe specific embodiments and not to be construed as limiting.

[0053] WD repeat domain 1 (WDR1): Also known as actin interaction protein 1 (AIP1), it is a highly conserved cytoskeleton-associated protein. It participates in regulating actin filament dynamics and actin-dependent cell biological processes, such as intercellular junction remodeling and cytoskeleton regulation, and plays an important role in cell division and migration.

[0054] Acute myeloid leukemia (AML): a malignant blood cancer originating in the bone marrow, characterized by the production of large numbers of immature white blood cells (called leukemia cells or AML cells). These immature cells cannot function properly and accumulate in large numbers, interfering with the production of normal blood cells.

[0055] Acute myeloid leukemia stem cells (AML LSCs): These are cells with self-renewal and multi-lineage differentiation capabilities, and they are the main source of acute myeloid leukemia. AML LSCs are resistant to conventional chemotherapy drugs, which is a significant cause of disease relapse and drug resistance. There are different interpretations among those skilled in the art regarding the definition of AML LSCs. For example, in this invention, human leukemia stem cells are defined as the Lineage-CD34+CD38- population, and mouse leukemia stem cells are defined as the c-Kit+Gr1- population. Others may define L-GMP as a leukemia stem cell population.

[0056] Cellular activity inhibition: This technique reduces the number of cancer cells by suppressing cell growth and proliferation, thereby achieving a therapeutic effect. Cellular activity inhibition is typically achieved by inducing apoptosis or preventing cell division.

[0057] Apoptosis: a programmed cell death process, an ordered suicide procedure executed by cells in response to specific stimuli. Apoptosis is an important mechanism for maintaining homeostasis in normal organisms.

[0058] Gambogic amide, also known as flavonoid amide, glycyrrhizin amide, etc., has the CAS number 286935-60-2 and its structural formula is shown in Formula I. Gambogic amide is a potent, selective, high-affinity TrkA receptor agonist that selectively binds to TrkA but not to TrkB or TrkC, and strongly induces its tyrosine phosphorylation and downstream signaling activation, including Akt and MAPKs; it effectively inhibits cyclin A1 expression and blocks K562 cell proliferation; and in a mouse model of transient middle cerebral artery occlusion following stroke, it reduces fumarin-induced neuronal death and infarct volume.

[0059]

[0060] I.

[0061] Morellic acid, also known as mollic acid, is a pyranone-type polyketide compound isolated from plants of the genus Garcinia (such as mangosteen husks). It belongs to the polycyclic polyisoprenylated phloroglucinol derivatives. The molecular formula of morellic acid is C1. 28 H 32O6, with the structural formula shown in Formula II, exhibits its main biological activity in the efficient inhibition of the STAT3 signaling pathway. By blocking its phosphorylation, it induces apoptosis in cancer cells and demonstrates significant anti-proliferative effects in various human cancer models (such as breast cancer, lung cancer, and leukemia) in vitro. Furthermore, it possesses antibacterial, antioxidant, and anti-inflammatory potential. Currently, this compound faces development challenges due to its low natural abundance, complex structure leading to difficulties in total synthesis, and insufficient in vivo pharmacokinetic data. However, its unique STAT3 targeting mechanism makes it an important lead compound template for anticancer drug development.

[0062]

[0063] II.

[0064] Flavopiridol hydrochloride, also known as avopiridol hydrochloride, is a semi-synthetic flavonoid molecule derived from the natural product of the Indian plant *Dysoxylum binectariferum*. The molecular formula of flavopiridol hydrochloride is C2. 21 H 20 ClNO5·HCl, with the structural formula shown in Formula III, is the first pan-cyclin-dependent kinase inhibitor to enter clinical trials. It primarily targets CDK1, CDK2, CDK4, CDK6, and CDK9, inhibiting the activity of these key kinases to induce cell cycle arrest (G1 / S or G2 / M phases) and downregulate short-lived pro-survival proteins (such as Mcl-1), thereby inducing apoptosis in cancer cells. This drug has undergone multiple clinical trials for the treatment of hematologic malignancies (such as acute and chronic leukemia and lymphoma) and solid tumors, showing particular potential for synergistic effects when used in combination with targeted therapies or chemotherapy. Its main challenges lie in its narrow therapeutic window, and common dose-limiting toxicities include severe diarrhea, cytokine release syndrome, and tumor lysis syndrome. Despite limited efficacy as a monotherapy, Flavopiridol, as a cornerstone of combination therapies and a pioneer of CDK inhibitors, has profoundly influenced the development of targeted cell cycle therapies.

[0065]

[0066] III.

[0067] Gambogic Acid, also known as gambogic acid, is a natural xanthones compound extracted from the dried resin (gamboge) of plants in the genus Garcinia (such as Garcinia hanburyi) in East Asia. The molecular formula of Gambogic Acid is C2. 38 H 44O8, with the structural formula shown in Formula IV, is a highly effective apoptosis inducer. Its core mechanism involves directly targeting multiple cancer-related proteins, including binding to and inhibiting client proteins of heat shock protein 90 (such as Bcl-2 and c-Myc), inhibiting the activity of topoisomerase IIα, and triggering ferroptosis by inducing reactive oxygen species (ROS) generation. This compound has demonstrated broad-spectrum anticancer activity against various solid tumors (such as liver cancer, lung cancer, and gastric cancer) and hematological malignancies in preclinical studies and has entered Phase II clinical trials in China for the treatment of various cancers. Its main challenges lie in its poor water solubility, rapid elimination in vivo, and certain dose-dependent cardiotoxicity. Current research is working to improve its pharmaceutical properties through structural modification, nanodelivery systems, and combination therapy strategies, aiming to make it a unique natural anticancer lead compound with a clear multi-target mechanism of action.

[0068]

[0069] IV.

[0070] Methylophiopogonanone B, also known as methyl ophiopogon dihydroisoflavone B, is a unique dihydroisoflavone natural product isolated from the tuberous root of the traditional Chinese medicine Ophiopogon japonicus. The molecular formula of Methylophiopogonanone B is C1. 19 H 18 O5, with the structural formula shown in Formula V, exhibits core pharmacological activities focused on cardiovascular protection and anti-inflammation. By significantly inhibiting the expression of intercellular adhesion molecule-1 in vascular endothelial cells, it effectively reduces leukocyte adhesion and inflammatory infiltration, thus demonstrating its potential to combat atherosclerosis and myocardial ischemia-reperfusion injury in experimental models. Furthermore, this compound also exhibits certain antioxidant and anti-apoptotic activities. Current research is primarily in the preclinical stage. Its low natural abundance and unclear in vivo metabolic properties are the main bottlenecks restricting its further development, making it a key research target for revealing the traditional pharmacodynamic material basis of Ophiopogon japonicus and developing lead compounds specific for cardiovascular and cerebrovascular diseases.

[0071]

[0072] V.

[0073] Example 1. Screening of small flavonoid compounds

[0074] Five hundred and eighteen flavonoid small molecule compounds at a concentration of 1 μM were selected and used to treat human CD34-positive AML cells KG-1a. After 72 hours of cell culture, cell viability was assessed, and OD450 was analyzed. The values ​​were then converted using Log2 to obtain the leukemia stem cell clearance score. The results showed that five compounds, including Gambogic amide, had an inhibitory effect on AML cell viability. Furthermore, based on the top five candidate compounds with lower scores, the results were validated in KG-1a and KASUMI-1 cells, revealing that Gambogic amide significantly inhibited CD34-positive AML cells. + For details on the cell viability of AML LSCs, please refer to [link / reference]. Figure 1 .

[0075] Example 2. The flavonoid small molecule compound Gambogic amide in human CD34 + Analysis of its role in AML cells

[0076] First, select human CD34. + AML cells KG-1a and KASUMI-1 were seeded in 96-well plates and treated with different concentrations of Gambogic amide or DMSO. After 72 hours of cell culture, CCK8 reagent was added, and after 2 hours of incubation, OD450 was measured, and IC50 analysis of the compounds was performed. Results are as follows: Figure 2 As shown in a, the IC50 of Gambogic amide is in the range of 0.28 μM to 0.6 μM.

[0077] Next, 40,000 KG-1a and KASUMI-1 cells were seeded into 96-well plates, and CD34 cells were treated with 0.2 μM, 0.4 μM, and 0.8 μM Gambogic amide, respectively. + AML cells were analyzed for OD450 after 72 hours. Results are as follows: Figure 2 As shown in the report, Gambogic amide can take effect after 20 hours and remains effective after six days.

[0078] Finally, collect 1×10 6 One KASUMI-1 cell was transplanted into NOG recipient mice irradiated with 2 Gy, and the mice were treated with 1 mg / kg Gambogic amide (n=5 per group). The mice's condition was observed, and the survival curves were calculated. The results are as follows: Figure 2 As shown in f, Gambogic amide can effectively inhibit the pathogenesis of AML in mice.

[0079] Example 3. Analysis of the role of the flavonoid small molecule compound Gambogic amide in mouse AML LSCs

[0080] First, 5×10 were collected. 5 MLL-AF9-positive mouse AML LSCs were seeded in 12-well plates and treated with 0.2 μM, 0.4 μM, and 0.8 μM Gambogic amide. Cells were harvested after 72 hours, and apoptosis levels were assessed using Annexin V and PI. Results are as follows: Figure 3 As shown in Figure a, the analysis showed that Gambogic amide significantly induced an increase in apoptosis levels in mouse AML LSCs.

[0081] Secondly, 1×10 were collected. 6 MLL-AF9 positive cells were transplanted into irradiated recipient mice to construct an AML mouse model. Two weeks later, mice were intraperitoneally injected with 1 mg / kg Gambogic amide five times, one day apart. Peripheral blood cells were collected at week five to analyze changes in the proportion of AML LSCs in the mice. The results are as follows: Figure 3 As shown in bc, Gambogic amide treatment significantly reduced the proportion of AML LSCs in mice.

[0082] Finally, the survival status of the mice was observed, and the survival curves were statistically analyzed. The results are as follows: Figure 3 As shown in d, Gambogic amide intervention can significantly delay the development of AML in mice.

[0083] Example 4. Toxicity analysis of the flavonoid small molecule compound Gambogic amide on normal hematopoietic stem cells.

[0084] First, normal human peripheral blood samples were selected, cleaved, washed once with PBS, resuspended in culture medium, and seeded into 12-well plates. 0.4 μM Gambogic amide was added for intervention. After 72 hours, cells were collected, labeled with the corresponding antibody, and analyzed by flow cytometry for human HSCs (Lineage-CD34). + CD38 - ) and living cells (DAPI) - The result is as follows: Figure 4 As shown in ab, Gambogic amide has very low toxicity to normal human cells and no significant effect on the proportion of HSCs.

[0085] Secondly, Gambogic amide was administered intraperitoneally at a dose of 1 mg / kg, for a total of three times, with each dose spaced one day apart. After 70 days, flow cytometry analysis was performed to analyze the proportion of bone marrow-derived HSCs and changes in mature cells. Results are as follows: Figure 4As shown in cf, after in vivo intervention with Gambogic amide, the proportions of mature cells and hematopoietic stem cells in mice did not change significantly, suggesting that Gambogic amide has very little toxicity to the hematopoietic system.

[0086] Example 5. Analysis of the induction of apoptosis by the flavonoid small molecule compound Gambogic amide through partial binding to the specific protein WDR1 in AML LSCs.

[0087] First, single-cell data from GSE116256 were selected to analyze the changes in WDR1 expression levels in AML LSCs and HSCs. The results are as follows: Figure 5 As shown in figure a, WDR1 is specifically highly expressed in AML LSCs. This result also suggests that Gambogicamide may target and bind to WDR1 to clear AML LSCs. Since WDR1 is expressed less in HSCs, Gambogic amide cannot bind sufficiently to induce apoptosis.

[0088] Secondly, select 1×10 7 KG-1a cells were seeded into 6-well plates, and 3 μM Gambogic amide was added. After 3 hours, the cells were collected, divided into 9 aliquots, and heated in a PCR instrument with gradually increasing temperature for 3 minutes. Cells were then collected to extract proteins for Western blot analysis. The results are shown below. Figure 5 As shown in b, WDR1 was more stable after Gambogic amide intervention, suggesting that Gambogic amide and WDR1 are bound together.

[0089] Finally, cells were seeded and either the F-actin promoter Jas or the F-actin depolymerization agent Gambogicamide was added. Changes in cell membrane potential and the effect of Gambogic amide on apoptosis levels were analyzed. Results are as follows: Figure 5 As shown in ce, Gambogic amide can bind to WDR1, depolymerize F-actin, reduce membrane potential, and lead to apoptosis.

[0090] Example 6. Comparison of the effects of Gambogic amide and the existing chemotherapy drug cytarabine on inhibiting AML LSCs;

[0091] 40,000 CD34 + AML cells, KG-1a or KASUMI-1, were seeded into 96-well plates, and 0.4 μM Gambogic amide or cytarabine was added. Cell viability was analyzed after 72 hours. Results are as follows: Figure 6 As shown, Gambogic amide can effectively inhibit CD34. + The activity of AML cells was significantly enhanced compared to cytarabine.

[0092] The above results indicate that the flavonoid compound Gambogic amide of this invention can effectively inhibit the activity of AML LSCs and induce an increase in apoptosis levels. It exhibits highly efficient scavenging effects on AML LSCs both in vivo and in vitro. Furthermore, Gambogic amide showed low toxicity to HSCs, which was correlated with low expression of WDR1 in HSCs. Figure 7 Compared with traditional cytarabine chemotherapy drugs, Gambogic amide has a strong ability to inhibit the activity of AML LSCs. In summary, the application of Gambogic amide in clearing AML LSCs has profound scientific research and clinical significance and important prospects for widespread application.

Claims

1. The use of flavonoid small molecule compounds or their solvates, optical isomers, polymorphs or pharmaceutically acceptable salts thereof in the preparation of drugs that regulate WDR1, characterized in that, The flavonoid small molecule compounds include any one or more of Gambogicamide, Morellitic acid, Flavopiridol hydrochloride, Gambogic Acid, and Methylophiopogonanone B; the structural formulas of each flavonoid small molecule compound are as follows: 。 2. The application according to claim 1, characterized in that, The flavonoid small molecule compound is Gambogicamide.

3. The application according to claim 1, characterized in that, The regulation includes positive regulation and / or negative regulation.

4. The use of flavonoid small molecule compounds or their solvates, optical isomers, polymorphs or pharmaceutically acceptable salts thereof in the preparation of medicaments for the treatment of leukemia, characterized in that, The flavonoid small molecule compounds include any one or more of Gambogic amide, Morellitic acid, Flavopiridol hydrochloride, Gambogic Acid, and Methylophiopogonanone B.

5. The application according to claim 4, characterized in that, The treatment comprises administration of 0.2 μM to 0.8 μM flavonoids; wherein, before treatment, the subject or experimental animal has a baseline level of leukemia stem cell viability of more than 95%, a stem cell proportion of more than 8%, a late apoptosis baseline level of less than 25%, a cell differentiation baseline level of less than 40%, and a survival time of ≤48 days; after treatment with the flavonoid small molecule compound, the subject or experimental animal has a stem cell viability of less than 60%, a stem cell proportion of less than 2%, a late apoptosis level of more than 55%, a cell differentiation baseline level of more than 60%, and a survival time of more than 48 days.

6. The application according to claim 4, characterized in that, The leukemia includes acute myeloid leukemia and relapsed / refractory acute myeloid leukemia; the acute myeloid leukemia is caused by WDR1-specific high expression.

7. The application according to claim 4, characterized in that, The use of the flavonoids or their solvates, optical isomers, polymorphs or pharmaceutically acceptable salts thereof in the preparation of medicaments for delaying the progression of leukemia, clearing AML LSCs, inhibiting the cellular activity of AML LSCs and / or inducing apoptosis of AML LSCs.

8. The use of flavonoid small molecule compounds or their solvates, optical isomers, polymorphs or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating diseases caused by WDR1, characterized in that, The flavonoid small molecule compounds include any one or more of Gambogic amide, Morellitic acid, Flavopiridol hydrochloride, Gambogic Acid, and Methylophiopogonanone B.

9. The application according to claim 8, characterized in that, The diseases caused by WDR1 include acute myeloid leukemia and refractory relapsed leukemia.

10. A pharmaceutical composition for the prevention or treatment of diseases caused by WDR1, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of a small flavonoid compound or its solvate, optical isomer, polymorph or pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient; the small flavonoid compound includes one or more of Gambogicamide, Morellitic acid, Flavopiridol hydrochloride, Gambogic Acid, and Methylophiopogonanone B.

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