Small molecule for inhibiting activity of REXO4 digestion R-loop enzyme and application of small molecule in preparation of tumor drugs

Abstract

The invention relates to the technical field of biological medicines, and discloses a small molecule for inhibiting the activity of REXO4 digestion R-loop enzyme and application of the small molecule in preparation of tumor medicines. The obtained small molecule can inhibit the activity of REXO4 digestion R-loop enzyme, so that the content of an R-loop structure in a tumor is increased, and the aim of treating the tumor is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a small molecule that inhibits the activity of REXO4 enzyme that digests R-loop and an application thereof in the preparation of tumor drugs. Background Art

[0002] R-loop structure is a structure that often appears during RNA transcription, consisting of a free DNA chain and an RNA-DNA hybrid chain. There are high-frequency transcription and replication events in dividing cells, especially tumor cells. During RNA transcription, when RNA polymerase encounters obstacles, R-loop structure may be formed.

[0003] Although R-loop structures can serve as important regulators under physiological conditions, such as regulating gene expression, regulating the occurrence of B cell CSR, regulating T cell receptor reprogramming, etc., the abnormal accumulation of R-loop structures can block transcription or expose single-stranded DNA to nucleases. In the S phase of the cell cycle, transcription-replication collisions occur between the DNA replication fork and the transcription complex, which is the most common situation of R-loop structure-mediated DNA damage. These collisions can lead to replication arrest or even the collapse of the replication fork, which in turn leads to DNA double-strand breaks, posing a risk to genome stability.

[0004] To mitigate the potential toxic consequences of R-loop structures, cells have evolved a variety of regulatory mechanisms to prevent their formation or eliminate them. For example, the endonuclease RNase H1 (RNASEH1) can specifically degrade the RNA chain of the R-loop, allowing the DNA chain to reanneal. Tumor cells face the risk of DNA damage caused by high replication and high transcription, and replication stress is the main source of endogenous DNA damage in tumor cells. In tumor cells, if the accumulated transcription-replication collisions cannot be effectively alleviated, the tumor cells will enter a state of mitotic collapse, leading to cell death.

[0005] Therefore, targeted degradation of enzymes related to the R-loop structure has important biological and clinical significance for tumor treatment. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a small molecule that inhibits the activity of REXO4 enzyme to digest R-loop and its use in the preparation of tumor drugs. The small molecule obtained by the present invention can inhibit the activity of REXO4 enzyme, thereby promoting the accumulation of R-loop structure in tumors and achieving the purpose of treating tumors.

[0007] The present invention provides a small molecule for inhibiting the activity of REXO4 in digesting R-loop enzymes, wherein the structural formula of the small molecule is: .

[0008] The present invention also provides an application of the small molecule that inhibits the R-loop digestion enzyme activity of REXO4 in the preparation of a drug for treating tumors.

[0009] Furthermore, the tumor includes one or more of human head and neck squamous cell carcinoma and oral squamous cell carcinoma.

[0010] The present invention also provides an application of the small molecule that inhibits the R-loop digestion enzyme activity of REXO4 in the preparation of a drug for inhibiting the enzyme activity of REXO4.

[0011] The present invention also provides an application of the small molecule that inhibits the R-loop digestion enzyme activity of REXO4 in the preparation of a drug for promoting the accumulation of R-loop structures in tumor cells.

[0012] The embodiments of the present invention have the following technical effects: 1. The small molecule obtained in the present invention can inhibit the enzyme activity of REXO4 in digesting the R-loop structure, thereby promoting the accumulation of R-loop structures in tumors, achieving the purpose of treating tumors.

[0013] 2. The small molecule obtained by the method of the present invention can inhibit the enzyme activity of REXO4 in digesting the R-loop, enhance the level of R-loop structures in UM-SCC1 cells and UM-SCC25 cells, achieving the purpose of treating human head and neck squamous cell carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0015] Figure 1 is the mass spectrometry diagram of the small molecule in Example 1 of the present invention.

[0016] Figure 2 is the preparation process of the small molecule obtained in Example 1 of the present invention.

[0017] Figure 3 is the test result of Example 2 of the present invention, where Figure 3 A in it is the cleavage preference of REXO4 enzyme; Figure 3 B in it is a typical diagram and statistical chart of the in vitro exonuclease efficiency of REXO4 enzyme on the hybrid strand of R-loop; Figure 3 C in it is the Coomassie brilliant blue stained gel diagram of His-REXO4 protein.

[0018] Figure 4 These are the test results of Example 3 of the present invention, where Figure 4 in A, it is the cleavage efficiency of REXO4 on RNA in the RNA-DNA hybrid strand at different iR4 concentrations; Figure 4 in B, it is a statistical chart of the cleavage efficiency of REXO4 on RNA in the RNA-DNA hybrid strand at different iR4 concentrations.

[0019] Figure 5 These are the test results of Example 4 of the present invention, where Figure 5 in A, different doses of iR4 are used to treat UM-SCC1 cells, and GFP-dRH1 is detected; Figure 5 in B, it is a statistical chart of the relative fluorescence intensity of GFP in UM-SCC1 cells; Figure 5 in C, different doses of iR4 are used to treat UM-SCC25 cells, and GFP-dRH1 is detected; Figure 5 in D, it is a statistical chart of the relative fluorescence intensity of GFP in UM-SCC25 cells.

[0020] Figure 6 These are the test results of Example 5 of the present invention, where Figure 6 in A, GFP-dRH1 in UM-SCC1 cells that can express RnaseH1 protein and cannot express RnaseH1 protein is detected by treating with iR4 added and without iR4 added; Figure 6 in B, it is a statistical chart of the relative fluorescence intensity of GFP in UM-SCC1 cells; Figure 6 in C, GFP-dRH1 in UM-SCC25 cells that can express RnaseH1 protein and cannot express RnaseH1 protein is detected by treating with iR4 added and without iR4 added; Figure 6 in D, it is a statistical chart of the relative fluorescence intensity of GFP in UM-SCC25 cells.

[0021] Figure 7 These are the test results of Example 6 of the present invention, where Figure 7 in A, it is a mouse tumor-bearing model, typical tumor pictures and statistical charts; Figure 7 in B, it is GFP-dRH1 tissue immunofluorescence staining and statistical charts; Figure 7 in C, it is γH2AX tissue immunofluorescence staining and statistical charts; Figure 7 in D, it is Cxcl10 tissue immunofluorescence staining and statistical charts, Figure 7 in E, it is AcCasp3 tissue immunofluorescence staining and statistical charts. Detailed implementation manners

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative work fall within the scope of protection of the present invention.

[0023] In a first aspect, in some embodiments of the present invention, a small molecule that inhibits the enzymatic activity of REXO4 in digesting R-loop is provided, and the structural formula of the small molecule is: .

[0024] In a second aspect, in some embodiments of the present invention, an application of the small molecule that inhibits the enzymatic activity of REXO4 in digesting R-loop in the preparation of a drug for treating tumors is also provided.

[0025] In some embodiments, the tumors include one or more of human head and neck squamous cell carcinoma and oral squamous cell carcinoma.

[0026] In a third aspect, in some embodiments of the present invention, an application of the small molecule that inhibits the enzymatic activity of REXO4 in digesting R-loop in the preparation of a drug for inhibiting the enzymatic activity of REXO4 in digesting R-loop is also provided.

[0027] In a fourth aspect, in some embodiments of the present invention, an application of the small molecule that inhibits the enzymatic activity of REXO4 in digesting R-loop in the preparation of a drug for promoting the accumulation of R-loop structure in tumor cells is also provided.

[0028] The following is elaborated with specific examples: Example 1: , denoted as: iR4.

[0029] The preparation of the product in Example 1 was entrusted to WuXi AppTec Co., Ltd. in Tianjin for preparation, and its preparation process is as Figure 2 shown.

[0030] NMR data: 1HNMR: EC29906-10-P1N (400 MHz, DMSO- d 6) δ ppm = 10.36 (s, 1H), 8.38(d, J = 7.6 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H), 7.92 (d, J= 7.6 Hz, 1H), 7.76 - 7.64(m, 4H), 7.54 - 7.42 (m, 4H), 7.34 - 7.28 (m, 2H), 7.26 - 7.19 (m, 3H), 5.61 (s, 2H), 4.47 (s, 2H).

[0031] The mass spectrometry data of the product of Example 1 are as Figure 1 shown.

[0032] Example 2: To verify that REXO4 enzyme can specifically degrade the RNA in R-loop structure, different substrates (such as ssRNA, RNA-DNA, etc.) were used to detect the cleavage preference of REXO4 enzyme. The results are as Figure 3 shown in A. It can be found that the in vitro enzyme activity experiment verified that REXO4 enzyme can specifically cleave the RNA in RNA-DNA hybrid strand. In Figure 3 B, it was further verified that REXO4 achieved the purpose of eliminating R-loop structure by degrading the RNA in the simulated human R-loop structure. Figure 3 C in is the coomassie staining map of REXO4 protein used in the in vitro enzyme activity detection experiment.

[0033] Example 3: To verify that the small molecule inhibitor iR4 (C 28 H 21 N5OS) can inhibit the exonuclease activity of REXO4, the results are as Figure 4 shown. In Figure 4 A and B, it can be found that the inhibition of the enzyme activity of REXO4 by iR4 in digesting R-loop shows concentration dependence. As the concentration of iR4 increases, the inhibition of the enzyme activity of REXO4 by the small molecule becomes more significant, so the degradation rate of RNA in RNA-DNA hybrid strand decreases. In addition, the half inhibitory concentration IC50 of iR4 on the enzyme activity of REXO4 is about 1 μM.

[0034] Example 4: To further verify that iR4 can inhibit the enzyme activity of REXO4 at the cellular level, it was verified by staining tumor cells with GFP-dRH1. The results are as Figure 5 shown. UM-SCC1 cells and UM-SCC25 cells (both UM-SCC1 cells and UM-SCC25 cells are human head and neck squamous cell carcinoma cells) were treated with different concentrations of iR4. Figure 5 In A - B and Figure 5Both C and D show that iR4 can specifically enhance the accumulation of R-loop structures in tumor cells. Among them, GFP-dRH1 is a recombinant protein expressed and purified in BL21 Escherichia coli, which consists of a GFP tag and an RNH1 D210N mutant, and is mainly used to study the regulatory mechanism of RNA-DNA hybrids (R-loops). Therefore, the R-loop structure in tumor cells can be labeled after cell staining. In Figure 5 it can be found that as the concentration of iR4 increases, the level of R-loop structures in UM-SCC1 cells and UM-SCC25 cells first increases and then levels off. Therefore, the further optimized concentration of iR4 is 5 μM.

[0035] Example 5: To further verify the specificity of iR4 targeting the REXO4 enzyme, lentiviruses containing the pLVX-Tet-On-Advanced (Neo) expression plasmid (purchased from Guangzhou Youbao Biotechnology Co., Ltd., product number: VT1467) were used to infect SCC1 cells and SCC25 cells, and stable strains SCC1-TR and SCC25-TR were obtained through screening with G418 Geneticin. The stable strains after G418 Geneticin screening were respectively infected with lentiviruses carrying the pLVX-Tight-puro-NLS-RNaseH1-V5-FLAG plasmid (Clontech, product number: 632184), and stable cell lines that could induce the expression of ribonuclease H1 (RnaseH1) by doxycycline (Dox) were obtained through puromycin screening. Among them, RnaseH1 is a type of endonuclease that specifically degrades RNA in RNA-DNA hybrid strands. Figure 6 Among them, a solution with iR4 was used as the experimental group (iR4 was dissolved in 10% v / v DMSO, 40% v / v PEG300, 5% v / v TWEEN80, and the balance was PBS buffer solution, with a concentration of 5 μM), denoted as: iR4; at the same time, a solution without iR4 was used as the control group (10% v / v DMSO, 40% v / v PEG300, 5% v / v TWEEN80, and the balance was PBS buffer solution), denoted as: Vehicle. It can be in Figure 6It was found that in a stable cell line capable of expressing ribonuclease H1 (i.e., denoted as "Dox+") and a stable cell line unable to express RnaseH1 (i.e., denoted as "Dox-"), when iR4 (i.e., denoted as "+") was added, the fluorescence intensity of GFP in SCC1 cells capable of expressing RnaseH1 was significantly lower than that in SCC1 cells unable to express RnaseH1, and the fluorescence intensity of GFP in SCC1 cells unable to express RnaseH1 was significantly higher than that in SCC1 cells treated with a solution without iR4 (i.e., denoted as "-"). In SCC1 cells treated with a solution without iR4, the fluorescence intensity of GFP did not change, and the same result was verified in SCC25 cells. It can be seen that iR4 induces the accumulation of R-loop structures by specifically targeting REXO4, and part of the accumulated R-loop structures are eliminated by RnaseH1 induced by Dox.

[0036] Example 6: An animal model was established. 16 6-week-old C57BL / 6 mice (fully immunocompetent mice) were randomly divided into two groups. Each mouse was subcutaneously injected with 2.5×10 6 MOC1 cells (oral squamous cell carcinoma cells). Five days after injection, the largest tumor in the mice grew to approximately 100 mm 3 . The two groups of mice were treated with drugs every two days. The experimental group was injected with a solution containing iR4 at a dose of 10 mg / kg (the dose of iR4 was calculated according to the body weight and injection volume of the mice, and then iR4 was dispersed in 100 μL of solvent, which was 10% v / v DMSO, 40% v / v PEG300, 5% v / v TWEEN80, and the balance was PBS buffer solution), denoted as iR4; the other group of mice was injected with the same volume of solvent (10% v / v DMSO, 40% v / v PEG300, 5% v / v TWEEN80, and the balance was PBS buffer solution) at the same time point as the control group, denoted as Vehicle. Until 36 days after feeding the mice, the tumors and other indicators of the mice were detected. Figure 7 In A, it is a physical picture of the tumor cells of the mice 36 days after feeding the mice. It can be found that the tumors of the mice injected with iR4 were significantly smaller than those of the control group. The tumor cells were stained. In Figure 7 In B, it is the staining results of the cell nucleus of the tumor cells, the specific marker of head and neck squamous carcinoma cells, cytokeratin 14, and GFP-dRH1. Among them, K14 exists in the tumor cells, which can verify that both the control group and the iR4 group formed tumor cells. GFP-dRH1 staining was used to stain the R-loop structures in the tumor cells. In Figure 7 In B, it can be found that the content of R-loop structures in the tumor cells of the experimental group increased. It can be seen that iR4 can promote the accumulation of tumor R-loop structures. In Figure 7In C, γH2AX protein in tumor cells was stained. The γH2AX protein can reflect the DNA damage situation of tumor cells. In Figure 7 In C, it can be found that the DNA damage of tumor cells in the experimental group was significantly higher than that in the control group. It can be seen that iR4 can promote DNA damage of tumor cells and cause genomic instability. When the DNA of tumor cells is damaged, more cytoplasmic DNA will be produced. The cytoplasmic DNA can activate the expression of type I interferon genes. The production of type I interferon can recruit anti-tumor related immune cells to achieve the purpose of killing tumor cells. And chemokine Cxcl10 is one of the proteins in the signal pathway of type I interferon gene expression. It can be found in Figure 7 In D that the expression level of Cxcl10 in the experimental group was significantly higher than that in the control group. It can be seen that iR4 in the present invention can enhance the expression of chemokines of type I interferon genes, thereby realizing the recruitment of anti-tumor related immune cells. Staining was performed on the apoptosis-related marker AcCasp3 of tumor cells, and the results are as shown in Figure 7 In E. AcCasp3 can promote the apoptosis of tumor cells. It can be found that the fluorescence intensity of AcCasp3 in the experimental group was significantly higher than that in the control group. It can be seen that iR4 in the present invention can promote the apoptosis of tumor cells through anti-tumor immunity.

[0037] In summary, the small molecule obtained in the present invention can inhibit the activity of REXO4 to degrade R-loop, thereby promoting the accumulation of R-loop structures in tumors and achieving the purpose of treating tumors.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A small molecule that inhibits the activity of REXO4 in digesting R-loop, characterized in that: The structural formula of the small molecule is: 。 2. Use of the small molecule that inhibits the activity of REXO4 enzyme that digests R-loop according to claim 1 in the preparation of drugs for treating tumors.

3. The use according to claim 2, characterized in that: The tumor includes one or more of human head and neck squamous cell carcinoma and oral squamous cell carcinoma.

4. Use of the small molecule for inhibiting the REXO4 enzyme activity for clearing R-loop according to claim 1 in the preparation of a drug for inhibiting the REXO4 enzyme activity for clearing R-loop.

5. Use of the small molecule that inhibits the R-loop digestion enzyme activity of REXO4 as claimed in claim 1 in the preparation of a drug that promotes the accumulation of R-loop structures in tumor cells.

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