Use of chrysophanol in preparation of anti-tumor drugs targeting IKZF3

Abstract

The present application relates to the application of moracin in the preparation of anti-tumor drugs targeting IKZF3, by reducing and / or removing the phosphorylation level of IKZF3, making IKZF3 form a high-affinity homodimer, and remodeling the IKZF3 dimer as a "super suppressor" to induce cell apoptosis. Compared with the prior art, the present application discloses for the first time that the phosphorylation of IKZF3 can be specifically removed by a small molecule drug moracin, which can be converted from a "cancer-promoting factor" to a "lethal suppressor", without inducing protein degradation, and overcoming the drug resistance of lenalidomide / pomalidomide and other anti-tumor immunomodulatory drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of sanguisorbin in the preparation of anti-tumor drugs targeting IKZF3. Background Technology

[0002] Hematologic malignancies such as multiple myeloma (MM) and diffuse large B-cell lymphoma (DLBCL) are highly dependent on a specific transcription factor network to maintain their survival and proliferation, a phenomenon known as "transcriptional addiction." Among these, Ikaros family zinc finger proteins, particularly Aiolos (IKZF3), are key regulators of B-cell lineage development and function. In malignant tumors, IKZF3 is often hijacked to maintain high expression of proto-oncogenes such as MYC and IRF4, thereby promoting tumor cell survival. Currently, most drugs targeting IKZF3 are immunomodulatory drugs (IMiDs), such as thalidomide, lenalidomide, and pomalidomide. These drugs act as "molecular glues," binding to the E3 ubiquitin ligase Cereblon (CRBN), altering its substrate specificity, recruiting IKZF1 and IKZF3 for ubiquitination, and ultimately degrading them via the proteasome pathway, thus exerting an anti-lymphoma effect. Since these drugs all exert their effects through the CRBN-dependent ubiquitination degradation pathway, they generally suffer from defects such as acquired resistance, poor target selectivity, and insufficient central penetration, thus limiting their clinical benefits.

[0003] Despite the great clinical success of "degrading agents" such as linalidomide, the following technical problems still urgently need to be solved: (1) Acquired drug resistance: The efficacy of existing technologies is highly dependent on the expression and function of the E3 ligase CRBN. Clinically, it is common for drugs to fail to recruit IKZF3 for degradation due to CRBN gene mutations, downregulation, or deletion, resulting in severe drug resistance.

[0004] (2) Limitations of loss of function: IKZF3 inherently possesses transcriptional repressive potential (e.g., recruiting NuRD complexes). Existing technologies, by completely eliminating (degrading) the protein, while blocking its pro-survival function, also deprive it of the opportunity to be transformed into a "tumor suppressor".

[0005] (3) Off-target toxicity: Systemic protein degradation may lead to the loss of physiological IKZF1 / 3 in non-tumor cells (such as T cells and neutrophils), causing side effects such as neutropenia. Summary of the Invention

[0006] The purpose of this invention is to overcome the above-mentioned defects in the anti-tumor effect exerted through the CRBN-dependent ubiquitination degradation pathway, thereby providing the application of sanguisorbin in the preparation of anti-tumor drugs targeting IKZF3.

[0007] The objective of this invention can be achieved through the following technical solutions: One of the technical solutions of the present invention is to provide the application of sanguisorbin in the preparation of an anti-tumor drug targeting IKZF3, wherein the anti-tumor drug is a drug that reduces and / or removes the phosphorylation level of IKZF3.

[0008] In some specific embodiments, the antitumor drug is a drug that reduces and / or removes the phosphorylation level at any of the following sites (1) to (4) in IKZF3.

[0009] (1) N-terminal Tyr29 (Y29) site; (2) C-terminal Ser378 (S378) site; (3) C-terminal Thr389 (T389) site; (4) N-terminal Ser17 (S17) site.

[0010] In some specific embodiments, the antitumor drug is a drug that increases immune-related genes and endoplasmic reticulum stress-related genes by reducing and / or removing IKZF3 phosphorylation levels.

[0011] In some specific embodiments, the immune-related genes include IFNG and CCR7.

[0012] In some specific embodiments, the endoplasmic reticulum stress-related genes include XBP1, DDIT3, ATF4, and PRDM1.

[0013] In some specific embodiments, the antitumor drug is an anti-lymphoma drug.

[0014] In some specific embodiments, the lymphoma targeted by the anti-lymphoma drug is B-cell non-Hodgkin lymphoma.

[0015] In some specific embodiments, the B-cell non-Hodgkin lymphoma is selected as diffuse large B-cell lymphoma, and the cell line of the diffuse large B-cell lymphoma is selected from any one or more of SUDHL-4, RIVA, and RL4RH.

[0016] The second technical solution of the present invention is to provide a method for screening anti-tumor drugs, including detecting whether the test substance reduces and / or removes the phosphorylation level of IKZF3 in tumor tissue, and the candidate drugs obtained by screening are anti-tumor drugs.

[0017] In some specific embodiments, the tumor is a lymphoma, preferably a B-cell non-Hodgkin lymphoma, such as diffuse large B-cell lymphoma, wherein the cell line of the diffuse large B-cell lymphoma is selected from any one or more of SUDHL-4, RIVA, and RL4RH.

[0018] The third technical solution of the present invention is to provide an antitumor drug that targets and reduces and / or removes IKZF3 phosphorylation, wherein the antitumor drug includes sanguisorbin and a pharmaceutically acceptable carrier.

[0019] In some specific embodiments, the tumor is lymphoma, preferably B-cell non-Hodgkin lymphoma, such as diffuse large B-cell lymphoma, wherein the cell line of the diffuse large B-cell lymphoma is selected from any one or more of SUDHL-4, RIVA, and RL4RH. Morusin is a naturally derived small molecule compound. Existing research only involves preliminary reports on its anti-inflammatory and partial tumor cytotoxic effects. There are no reports on the use of Morusin as an IKZF3 targeting modulator in the preparation of anti-tumor drugs, nor is there any publicly available technology revealing its specific action on IKZF3 for lymphoma treatment. This invention creatively develops a novel small molecule drug that targets IKZF3 function in a non-degradation mode, which has significant clinical value and market prospects.

[0020] This invention creatively discovers that sanguisorbin can directly bind to the IKZF3 protein and reshape its function through a unique "phosphorylation switch" mechanism, rather than inducing its degradation.

[0021] Sangxin induces a sharp decrease in phosphorylation levels at the N-terminal Tyr29 (Y29), C-terminal Ser378 (S378), C-terminal Thr389 (T389), and N-terminal Ser17 (S17) sites of IKZF3, leading to the formation of a high-affinity homodimer. This remodeled IKZF3 dimer acts as a "hyper-repressor," tightly binding to the promoter regions of oncogenes such as MYC or IRF4, recruiting co-repressive complexes (such as NuRD), forcibly shutting down survival signals, and thereby inducing apoptosis.

[0022] Compared with the prior art, the present invention has the following advantages: (1) Pioneering “non-degradation” targeting mode: This invention reveals for the first time that by specifically removing the phosphorylation of IKZF3 through the small molecule drug Sangxinsu, it can be transformed from a “cancer-promoting factor” into a “lethal inhibitory factor” without inducing protein degradation.

[0023] (2) Unique “Trojan horse” mechanism: Unlike existing drugs, the drug Sangxinsu of this invention must rely on the presence of IKZF3 protein to exert its killing effect (IKZF3 knockout leads to drug resistance), which proves that the drug uses IKZF3 in cancer cells as a “killer”.

[0024] (3) Overcoming resistance to antitumor immunomodulatory drugs such as lenalidomide / pomalidomide: Since the mechanism of action does not depend on the CRBN-ubiquitination pathway, the drug Sangxinsu of this invention can effectively treat patients who are resistant to lenalidomide / pomalidomide (such as those with CRBN mutations or low expression), and has great clinical substitution value.

[0025] (4) Higher selectivity and safety: The drug Sangxinsu of the present invention modifies only the specific phosphorylation site of IKZF3, rather than clearing the protein systemically. Compared with traditional CRBN-dependent molecular glue drugs such as lenalidomide, it may reduce immune system side effects (such as neutropenia) caused by the complete absence of IKZF1 / 3. Attached Figure Description

[0026] Figure 1 The effect of biotin-labeled morusin on lymphoma cell viability.

[0027] Figure 2 This is a graph showing the results of a pull-down experiment demonstrating the specific binding of Morusin to IKZF3 protein. The first column (Input) represents the total protein loading control. The second column (Biotin) represents the free biotin control group. The third column (Bio-Morusin) represents biotin-labeled Morusin treatment. The fourth column (Bio-Morusin + Morusin) represents the competitive binding group, where an excess of unlabeled Morusin was added concurrently with Bio-Morusin treatment.

[0028] Figure 3 The figure shows the results of the Cellular Thermal Displacement Analysis (CETSA) experiment to verify the stabilization of IKZF3 protein by Morusin in cells.

[0029] Figure 4 The binding kinetics of Morusin and IKZF3 protein were determined using surface plasmon resonance (SPR) experiments.

[0030] Figure 5 To analyze the effect of Morusin on the phosphorylation modification of IKZF3 protein using phosphorylated proteomics.

[0031] Figure 6To analyze the effects of Morusin on the expression of downstream and related functional genes of IKZF3 using mRNA sequencing (mRNA-seq).

[0032] Figure 7 The effects of IKZF3 gene knockout (CRISPR / Cas9) on cell survival, cell cycle and apoptosis of RL4RH cells treated with Morusin. Detailed Implementation

[0033] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0035] Unless otherwise specified, the materials and processes used in the following embodiments or examples are conventional materials and processes employed in the art to achieve the corresponding functions.

[0036] Example 1: Verification of the direct physical binding of sanguisorbin to the target protein IKZF3 This embodiment comprehensively demonstrates, through a variety of complementary biophysical and biochemical experiments, from in vitro affinity to intracellular stability, that Morusin directly targets and binds to IKZF3.

[0037] 1. Under four treatment conditions—Vehicle (solvent control), Biotin (biotin control), Morusin (unlabeled Morusin), and Biotin-Morusin (biotin-labeled Morusin)—RL4RH cells (1x10⁻¹⁰) were tested. 4 Dose-response curve of cell viability as a function of drug concentration (Log [Concentration]).

[0038] like Figure 1 The results showed that Biotin-Morusin had a similar cell viability inhibition effect to unlabeled Morusin, indicating that biotin labeling did not significantly affect the biological activity of Morusin and could be used for subsequent target capture experiments.

[0039] 2. Pull-down experiment Pull-down: Biotin-labeled morusin (10 μM) was reacted with two diffuse large B-cell lymphoma cell lines (RL4RH and RIVA, 5 × 10⁻⁶ cells each). 5 The cells were incubated with lysate and the complex was enriched using streptavidin magnetic beads.

[0040] like Figure 2 As shown, the third column shows that the IKZF3 protein band can be clearly detected after Bio-Morusin treatment, indicating that Bio-Morusin can specifically capture IKZF3. The fourth column shows that when an excess of unlabeled Morusin is added during Bio-Morusin treatment, the IKZF3 band is significantly weakened, proving that the two have competitive binding, further verifying the specific interaction between Morusin and IKZF3.

[0041] The above experiments confirmed that there is a physical interaction between sanguisorbin and IKZF3.

[0042] 3. Surface Plasmon Resonance (SPR) Analysis: Using the Biacore system, purified recombinant human IKZF3 protein (OriGene, RC207547) was immobilized on the surface of a CM5 sensor chip via amine coupling at an immobilization level of approximately 2000 RU. The binding and dissociation processes of SPR with the immobilized IKZF3 protein were monitored in real time by passing different concentration gradients (5 μM, 2.5 μM, 1.25 μM, 0.625 μM, 0.3125 μM, 0.15625 μM) of sanguisorbin solution, and the binding kinetic parameters were determined.

[0043] like Figure 4 The SPR sensor plots showed that the binding of sanguisorbin to IKZF3 protein was concentration-dependent and exhibited slow dissociation, demonstrating typical specific binding characteristics. The equilibrium dissociation constant (Kd) of sanguisorbin to IKZF3 protein was calculated to be 4.159 × 10⁻⁶. -6 M indicates that the two have a moderate affinity in vitro, further verifying the direct interaction between sanguisorbin and IKZF3, that is, the direct binding between sanguisorbin and IKZF3 with high affinity, which is the material basis for its potent pharmacological activity.

[0044] 4. Cell thermal displacement analysis (CETSA): In three diffuse large B-cell lymphoma cell lines (SUDHL-4, RIVA, and RL4RH), 5×10⁻⁶ cells were used for each cell line. 5 Add 10 μM sangin or DMSO to cells and perform heat lysis treatment at different gradients (e.g., 37-70°C). After lysis, the amount of soluble IKZF3 protein in the supernatant is detected by Western Blot.

[0045] like Figure 3 The results showed that, compared with the DMSO group, the thermal denaturation curve of IKZF3 protein in the sangxin treatment group shifted significantly to the right and the melting temperature increased significantly, indicating that the thermal stability of IKZF3 protein was significantly improved. The band intensity at the same temperature was significantly higher than that in the control group, indicating that sangxin can directly bind to and stabilize IKZF3 protein in cells, significantly increasing the thermal stability of the target protein, which ruled out the possibility of "false positive binding".

[0046] Example 2: Regulation (rather than degradation) of IKZF3 phosphorylation levels by Morusin (1) Treat RIVA and RL4RH cells (5×10⁶ cells each) with sine (10 μM). 5 (1 cell) 24 hours. The relative abundance changes of each phosphorylation site on the IKZF3 protein after treatment with sangxin were detected by quantitative phosphorylated proteomics (DMSO treatment group as control).

[0047] The results are as follows Figure 5 As shown, compared with the DMSO control group, the brightness of the IKZF3 protein band did not change significantly, indicating that no degradation occurred. In RIVA cells, the phosphorylation level of the C-terminal Ser378 / Thr389 (Log2FC=-9.40) of IKZF3 was significantly downregulated; in RL4RH cells, the phosphorylation levels of the N-terminal Ser17 (Log2FC=-9.28) and Tyr29 (Log2FC=-11.98) of IKZF3 were significantly downregulated. This indicates that sangin regulates IKZF3 function through dephosphorylation modification, and the dephosphorylation of the C-terminal S378 / T389 is closely related to IKZF3 dimerization and functional activation.

[0048] Unlike existing drugs such as lenalidomide, which cause rapid degradation of the IKZF3 protein, treatment with Morusin did not significantly reduce the abundance of IKZF3 protein. Instead, it significantly downregulated the phosphorylation level of the key functional domain of IKZF3 (the C-terminal dimerization region). This result indicates that the present invention inhibits tumors through a novel "functional remodeling" mechanism (i.e., dephosphorylation) rather than a "protein degradation" mechanism.

[0049] (2) Treat RIVA and RL4RH cells (5×10⁶ cells each) with senna (10 μM). 5Cells were used for 24 hours, with the DMSO-treated group as a control. The fold change in mRNA expression of related genes after treatment with sangxin was detected by mRNA-seq technology (LogFC). The genes detected included internal reference genes (Actin, GAPDH), downstream target genes of IKZF3 (IRF4, MYC, etc.), immune-related genes (IFNG, CCR7), and endoplasmic reticulum stress-related genes (XBP1, DDIT3, ATF4, PRDM1).

[0050] like Figure 6 The results showed that in both cell lines, the expression of downstream oncogenes of IKZF3 (such as IRF4 and MYC) was not significantly upregulated or was suppressed, while the expression of immune and endoplasmic reticulum stress-related genes (such as IFNG, PRDM1, XBP1, CCR7, DDIT3, and ATF4) was significantly upregulated. This indicates that sangxin exerts its anti-tumor effect by regulating IKZF3 function and altering its downstream transcriptional networks, such as the endoplasmic reticulum stress pathway. The gene expression changes were consistent in both cell lines, indicating that sangxin has a broad-spectrum regulation of IKZF3 function, providing a molecular basis for its anti-lymphoma effect.

[0051] Example 3: In vitro antitumor activity IKZF3 knockout (KO) lymphoma cell lines were constructed using CRISPR / Cas9 technology. Wild-type (WT) and IKZF3-KO cells were treated with sangin, and the apoptosis rate was detected.

[0052] Using RL4RH cells as a model, the control group was transfected with an empty vector, and the experimental group was transfected with three sgRNAs targeting IKZF3 (SgIKZF3-1, SgIKZF3-2, and SgIKZF3-3). GAPDH was used as an internal control protein. Western blot was used to detect the efficiency of CRISPR / Cas9 knockout of the IKZF3 gene. Results are as follows: Figure 7 As shown in figure a, compared with the Vector control group, all three SgIKZF3 experimental groups were able to effectively and significantly downregulate / knock out the expression of IKZF3 protein.

[0053] Next, the effect of different concentrations of morusin treatment on the cell viability of RL4RH cells in each group was tested, with a cell number of 5 × 10⁶. 5 One. The result is as follows. Figure 7The dose-response curve shown in b has the horizontal axis representing the logarithm of Morusin concentration and the vertical axis representing the relative cell viability. Under Morusin treatment, the cell viability of each SgIKZF3 group was higher than that of the Vector control group, indicating that the expression level of IKZF3 directly affects the sensitivity of cells to Morusin. IKZF3-KO leads to cell resistance to the drug, further confirming that IKZF3 is a key target for Morusin to exert its effects.

[0054] Further analysis was conducted on the changes in the cell cycle of RL4RH cells in each group, and the results are as follows: Figure 7 As shown in Figure c, under Morusin intervention, compared with the Vector control group, knocking out the IKZF3 gene significantly altered the distribution ratio of the cell cycle (the figure shows a statistically significant change in the ratio of S phase and G2 / M phase, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001).

[0055] Finally, the apoptosis rate (%) of RL4RH cells in each group was measured after 24 hours of treatment with Morusin. The results are as follows: Figure 7 As shown in d, knockout of the IKZF3 gene significantly inhibited Morusin-induced apoptosis in RL4RH cells (****p<0.0001).

[0056] The above results confirm that the cytotoxicity of sancin depends on the presence of the IKZF3 protein, supporting the "functional reprogramming / Trojan horse" mechanism, that is, the drug achieves lethal toxicity by modifying IKZF3, rather than by eliminating it.

[0057] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of sanguisorbin in the preparation of antitumor drugs targeting IKZF3, characterized in that, The antitumor drug is a drug that reduces and / or removes the phosphorylation level of IKZF3.

2. The application according to claim 1, characterized in that, The antitumor drug is a drug that reduces and / or removes the phosphorylation level at any of the following sites (1) to (4) in IKZF3: (1) N-terminal Tyr29 site; (2) C-terminal Ser378 site; (3) C-terminal Thr389 site; (4) N-terminal Ser17 site.

3. The application according to claim 1, characterized in that, The antitumor drug is one that increases immune-related genes and endoplasmic reticulum stress-related genes by reducing and / or removing IKZF3 phosphorylation levels.

4. The application according to claim 3, characterized in that, The immune-related genes include IFNG and CCR7.

5. The application according to claim 3, characterized in that, The endoplasmic reticulum stress-related genes include XBP1, DDIT3, ATF4, and PRDM1.

6. The application according to claim 1, characterized in that, The anti-tumor drug is an anti-lymphoma drug.

7. The application according to claim 6, characterized in that, The lymphoma targeted by the anti-lymphoma drug is B-cell non-Hodgkin lymphoma.

8. The application according to claim 6, characterized in that, The B-cell non-Hodgkin lymphoma was selected as diffuse large B-cell lymphoma, and the cell line of the diffuse large B-cell lymphoma was selected from any one or more of SUDHL-4, RIVA, and RL4RH.

9. A method for screening antitumor drugs, characterized in that, This includes detecting whether the test substance reduces and / or removes the phosphorylation level of IKZF3 in tumor tissue, and the candidate drugs obtained through screening are anti-tumor drugs.

10. An antitumor drug that targets and reduces and / or removes IKZF3 phosphorylation, characterized in that, The antitumor drugs include sanguisorbin and pharmaceutically acceptable carriers.

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