New application of RTKs / RAS / RAF / c-Myc signaling pathway inhibitor

Abstract

The invention belongs to the field of biological medicine, and particularly relates to novel application of an RTKs / RAS / RAF / c-Myc signal channel inhibitor. The invention discloses application of an RTKs / RAS / RAF / c-Myc signal channel inhibitor as a ferroptosis inhibitor. The RTKs signal channel inhibitor comprises BMS-599626, Lapatinib, and Ripretinib, and is characterized in that the RTKs signal channel inhibitor comprises BMS-599626, Lapatinib, and Ripretinib. And the RAS / RAF signal channel inhibitor comprises Lonafarnib and AZ628 (Azosulfuron 628). The c-Myc signal channel inhibitor comprises 10074-G5 and 10058-F4, and the c-Myc signal channel inhibitor comprises 10074-G5 and 10058 The invention provides a new thought for application of the RTKs / RAS / RAF / c-Myc signal channel inhibitor as a novel ferroptosis inhibitor in treatment of injury-related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a new use of RTKs / RAS / RAF / c-Myc signaling pathway inhibitors. Background Art

[0002] Ferroptosis is a non-apoptotic cell death mechanism that depends on the presence of redox-active metallic iron. Intracellular free iron or iron-containing enzymes react with oxygen and lipids containing polyunsaturated fatty acids (PUFAs) to generate high levels of membrane lipid peroxides, which is a key feature that distinguishes ferroptosis from other forms of cell death. When these membrane lipid peroxides accumulate at high levels, they can be lethal to cells. Therefore, any cell that contains iron, oxygen, and PUFA lipids is at risk for ferroptosis. In response to this oxidative membrane damage and the occurrence of ferroptosis, cells have evolved powerful enzymatic defense mechanisms to protect membranes from oxidative damage. Ferroptosis is a physiological process that plays a role in maintaining homeostasis, especially in tumor suppression. At the same time, ferroptosis may also be activated under acute and chronic disease conditions. Ferroptosis is the main cause of cell death associated with ischemia-reperfusion injury, at least in part due to ischemia-induced oxidative stress. These findings suggest that ferroptosis inhibitors are potential therapeutic approaches for treating diseases associated with ischemic injury. The inhibition of ferroptosis can be achieved through several key control points, the most important of which is to directly block the lipid peroxidation process, because ferroptosis is driven by the peroxidation of specific lipids. Soon after the discovery of ferroptosis, the potential applications of the first inhibitors were reported, such as Ferrostatin-1 and Liproxstatin, which effectively inhibit the propagation of lipid peroxidation as free radical scavengers and show efficacy in models such as glutamate toxicity, renal injury and liver injury. Therefore, it is of great research significance to have a deep understanding of the nature of ferroptosis and its regulatory mechanism.

[0003] Receptor tyrosine kinases (RTKs) are a subclass of tyrosine kinases that are involved in mediating a series of complex biological functions between cells, including cell growth, motility, differentiation and metabolism. There are 58 known RTKs in humans, all of which have similar protein structures, including an extracellular ligand binding domain, a single transmembrane helix and an intracellular region, where the intracellular region contains a juxtamembrane domain, a tyrosine kinase domain (TKD) and a carboxyl terminus. The epidermal growth factor receptor (EGFR) is the first receptor tyrosine kinase discovered, consisting of EGFR1 (EGFR, HER1, c-erbB1), HER2 (EGFR2, c-erbB2), EGFR3 (c-erbB3, HER3) and EGFR4 (c-erbB4, HER4). Platelet-derived growth factor receptor (PDGFR) is mainly expressed by cells of mesenchymal origin, such as fibroblasts, pericytes and vascular smooth muscle cells, and stimulates their proliferation and motility. PDGFRA and PDGFRB are classic oncogenes that encode the receptor tyrosine kinases for PDGF.

[0004] RAS proteins are important components of the signaling pathways from cell surface receptors. In humans, there are three RAS genes that encode four different but highly homologous RAS proteins of approximately 21 kDa: HRAS, NRAS, KRAS4A, and KRAS4B. RAS encodes a GTPase that transmits signals from growth factor receptors to downstream signaling cascades such as serine / threonine RAF kinases.

[0005] Activation of the MAPK signaling pathway directly or indirectly regulates a large number of cytoplasmic and nuclear substrates, such as the proto-oncogene Myc, which is located at the intersection of many growth-promoting signal transduction pathways and the immediate early response genes downstream of many ligand-membrane receptor complexes. The MYC oncogene family includes c-Myc, MYCN (N-Myc) and MYCL (L-Myc), and these three homologous genes have similar functions. Myc oncoprotein belongs to the so-called "super transcription factor" family and may regulate the transcription of at least 15% of the entire genome. The main downstream of Myc includes factors involved in ribosome generation, protein translation, cell cycle progression and metabolism, coordinating a wide range of biological functions such as cell proliferation, differentiation, survival and immune surveillance.

[0006] Currently, there are no reports showing that RTKs inhibitors (BMS-599626, Lapatinib, Ripretinib), RAS / RAF inhibitors (Lonafarnib, AZ628) and c-Myc inhibitors (10074-G5, 10058-F4) can block ferroptosis. Summary of the invention

[0007] First purpose of the invention: In order to overcome the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide the use of RTKs / RAS / RAF / c-Myc signaling pathway inhibitors as ferroptosis inhibitors.

[0008] The second purpose of the present invention is to provide the use of RTKs / RAS / RAF / c-Myc signaling pathway inhibitors in the preparation of ferroptosis blocking drugs.

[0009] Another object of the present invention is to provide an inhibitor of the RTKs / RAS / RAF / c-Myc signaling pathway for use as an inhibitor of ferroptosis in the preparation of drugs for treating injury-related diseases.

[0010] Technical solution: In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention is as follows:

[0011] The RTKs signaling pathway inhibitors of the present invention include BMS-599626, Lapatinib and Ripretinib.

[0012] RAS / RAF signaling pathway inhibitors include Lonafarnib and AZ628.

[0013] The c-Myc signaling pathway inhibitors include 10074-G5 and 10058-F4.

[0014] Lapatinib is a small molecule tyrosine kinase inhibitor targeting EGFR and ErbB2, which inhibits autophosphorylation and downstream signaling activities by interfering with the binding of adenosine triphosphate to its tyrosine kinase domain. Similarly, BMS-599626 is also an inhibitor of EGFR receptor tyrosine kinase. Ripretinib binds to wild-type and mutant forms of KIT and PDGFR, preventing them from transitioning from an inactive conformation to an active conformation, thereby inactivating them.

[0015] Lonafarnib is a specific inhibitor of RAS, which inhibits farnesyl transferase and prevents farnesylation of RAS protein, making it unable to bind to the cell membrane and function. AZ628 is a specific inhibitor of RAF, which inhibits the activation of CRAF by binding to the ATP binding site of RAF kinase.

[0016] 10074-G5 and 10058-F4 are specific inhibitors of c-Myc, inhibiting the interaction between c-Myc and Max and the transcriptional activation of its target genes.

[0017] The present invention adds RTKs / RAS / RAF / c-Myc signaling pathway inhibitors to cells induced by Erastin, RSL3 and -Cys (cysteine-deficient medium), and finds that BMS-599626, Lapatinib, Ripretinib, Lonafarnib, AZ628, 10074-G5 and 10058-F4 can effectively inhibit ferroptosis and the accumulation of lipid peroxides, indicating that they can be used as novel ferroptosis inhibitors to provide new treatment strategies for diseases related to organ damage.

[0018] The present invention further provides the use of RTKs / RAS / RAF / c-Myc signaling pathway inhibitors in inhibiting the positive regulator of ferroptosis, ACSL4. ACSL4 is a member of the long-chain acyl-CoA synthetase family, which catalyzes the binding of long-chain polyunsaturated fatty acids (PUFA) to coenzyme A to generate phospholipid PUFA, thereby promoting ferroptosis. RTKs inhibitors (BMS-599626, Lapatinib and Ripretinib), RAS / RAF inhibitors (Lonafarnib and AZ628), and c-Myc inhibitors (10074-G5 and 10058-F4) can all effectively downregulate the expression of ACSL4, demonstrating its potential application in ferroptosis inhibition.

[0019] The present invention provides that RTKs / RAS / RAF inhibitors block ferroptosis by down-regulating the expression of ACSL4 mediated by c-Myc. RTKs inhibitors (BMS-599626, Lapatinib and Ripretinib) and RAS / RAF inhibitors (Lonafarnib and AZ628) can significantly reduce the expression levels of c-Myc and ACSL4 proteins and RNA.

[0020] The beneficial effects of the present invention are as follows: the present invention discloses that RTKs / RAS / RAF / c-Myc signaling pathway inhibitors (including BMS-599626, Lapatinib, Ripretinib, Lonafarnib, AZ628, 10074-G5 and 10058-F4) significantly inhibit ferroptosis, and achieve the effect by down-regulating the ferroptosis positive factor ACSL4. Since these inhibitors have high safety, they can be used as new ferroptosis inhibitors and have broad application prospects in subsequent ferroptosis injury disease research. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :The effect of RTKs inhibitors on cell ferroptosis was observed by fluorescence microscopy. In the figure, a) Erastin, b) -Cys, c) RSL3;

[0022] Figure 2 :Flow cytometry analysis of the effects of RTKs inhibitors on cell ferroptosis. In the figure, a) Erastin, b) -Cys, c) RSL3;

[0023] Figure 3 :Flow cytometry was used to detect the effects of RTKs inhibitors on lipid peroxides (Lipid ROS) in cells. In the figure, a) Erastin; b) -Cys; c) RSL3;

[0024] Figure 4 :The effect of RAS / RAF inhibitors on cell ferroptosis observed by fluorescence microscopy In the figure, a) Erastin, b) -Cys, c) RSL3;

[0025] Figure 5 :The effect of RAS / RAF inhibitors on cell ferroptosis by flow cytometry. In the figure, a) Erastin, b) -Cys, c) RSL3;

[0026] Figure 6 :Flow cytometry was used to detect the effect of RAS / RAF inhibitors on Lipid ROS in cells. In the figure, a) Erastin, b) -Cys, c) RSL3;

[0027] Figure 7 :The effect of c-Myc inhibitor on cell ferroptosis observed by fluorescence microscopy

[0028] a) Erastin, b) RSL3;

[0029] Figure 8 Flow cytometry shows the effect of c-Myc inhibitors on cell ferroptosis. In the figure, a) Erastin, b) -Cys, c) RSL3;

[0030] Fig. 9 :Flow cytometry was used to detect the effect of c-Myc inhibitor on Lipid ROS in cells. In the figure, a) Erastin, b) -Cys, c) RSL3;

[0031] Fig.10 : Western blotting analysis of ACSL4 protein expression after treatment with c-Myc inhibitors (10074-G5 and 10058-F4);

[0032] Fig.11 :Real-time fluorescence quantitative PCR analysis of the changes in RNA levels of ACSL4 after treatment with c-Myc inhibitors (10074-G5 and 10058-F4);

[0033] Fig.12: Western blotting analysis of c-Myc and ACSL4 protein expression after treatment with RTKs inhibitors BMS599626, Lapatinib and Ripretinib In the figure, a) BMS-599626, b) Lapatinib, c) Ripretinib;

[0034] Fig.13 : Real-time fluorescence quantitative PCR analysis of the changes in RNA levels of c-Myc and ACSL4 after treatment with RTKs inhibitors BMS-599626, Lapatinib and Ripretinib. In the figure, a) BMS-599626, b) Lapatinib, c) Ripretinib;

[0035] Fig.14 : Western blotting analysis of c-Myc and ACSL4 protein expression after treatment with RAS / RAF inhibitors Lonafarnib and AZ628. In the figure, a) Lonafarnib, b) AZ628;

[0036] Fig.15 : Real-time fluorescence quantitative PCR was used to analyze the changes in RNA levels of c-Myc and ACSL4 after treatment with RAS / RAF inhibitors BMS599626, Lapatinib and Ripretinib.

[0037] In the figure, a) Lonafarnib, b) AZ628. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments to provide a better understanding of the present invention.

[0039] Embodiment 1

[0040] Evaluation of ferroptosis in HT1080 cells induced by erastin, cystine deficiency, and RSL3

[0041] Sensitivity to ferroptosis induced by RTKs / RAS / RAF / c-Myc signaling pathway inhibitors, including BMS-599626, Lapatinib, Ripretinib, Lonafarnib, AZ628, 10074-G5, and 10058-F4.

[0042] HT1080 human fibrosarcoma cells were cultured in high-glucose DMEM medium, to which double antibodies containing 100 U / ml penicillin and 0.1 mg / ml streptomycin and 10% (v / v) inactivated fetal bovine serum were added. The cells were cultured in a cell culture incubator at 37°C, 5% carbon dioxide concentration and high humidity.

[0043] Prepare HT1080 cell suspension and count the cells at 1.2×10 per well. 5 The cells were plated in 12-well plates at a density of 10 cells / mL and cultured overnight at 37°C. Subsequently, Erastin (10 μM), RSL3 (0.2 μM) and -Cys culture medium were added to the cells, and RTKs / RAS / RAF / c-Myc signaling pathway inhibitors (including 10 μM BMS-599626, 10 μM Lapatinib, 10 μM Ripretinib, 10 μM Lonafarnib, 10 μM AZ628, 50 μM 10074-G5 and 50 μM 10058-F4) were added. After Erastin treatment for 16 h, RSL3 treatment for 5 h, and -Cys treatment for 24 h, propidium iodide PI (1 mg / ml) dye was added to the cells at a ratio of 1:1000, incubated at 37°C for 5 min, and cell ferroptosis was observed by fluorescence microscopy.

[0044] Figure 1 This is the result of fluorescence microscopy observation in Example 1. 10 μM BMS599626, 10 μM Lapatinib and 10 μM Ripretinib were added to HT1080 cells induced by Erastin (10 μM), RSL3 (0.2 μM) and -Cys culture medium. After PI staining, the addition of RTKs inhibitors inhibited cell ferroptosis compared with the control group.

[0045] After recording with a fluorescence microscope, the cell supernatant was discarded, the cells were washed twice with PBS, trypsin was added to digest the cells, and after terminating the digestion, the cells were collected in a 1.5 mL EP tube and centrifuged at 3000 rpm and 4°C for 5 min; the supernatant was discarded, and the cells were resuspended in a flow tube with PBS containing 1% serum and detected by flow cytometry.

[0046] Figure 2 This is the statistical result of cell death by flow cytometry in Example 1. Compared with the control group, the addition of RTKs inhibitor inhibited cell ferroptosis.

[0047] Flow cytometry was used to count the changes in intracellular Lipid ROS. After the cells were treated as indicated and ferroptosis inducers were added, when the cells began to undergo ferroptosis under a microscope, 1 μL of 5 mM BODIPY 581 / 591C11 was added to each 1 mL of culture medium, and 0.5 μL of 2 mM Calcein AM was added to each 1 mL of culture medium as a positive control. The mixture was mixed evenly and incubated in a 37°C cell culture incubator for 1 h. The cell supernatant was discarded, washed twice with PBS, and trypsin was added to digest the cells. After digestion was terminated, the cells were collected in a 1.5 mL EP tube and centrifuged at 3000 rpm and 4°C for 5 min. The supernatant was discarded, and the cells were resuspended in a flow tube with PBS containing 1% serum, and detected by flow cytometry.

[0048] Figure 3 This is the statistical result of the cell Lipid ROS content by flow cytometry in Example 1. Compared with the control group, the Lipid ROS content was significantly reduced after the addition of RTKs inhibitors.

[0049] Figure 4 This is the result of fluorescence microscopy observation in Example 1. 10 μM Lonafarnib and 10 μM AZ628 were added to HT1080 cells induced by Erastin (10 μM), RSL3 (0.2 μM) and -Cys medium. After PI staining, ferroptosis was blocked after the addition of RAS / RAF inhibitors compared with the control group.

[0050] Figure 5 This is the statistical result of cell death by flow cytometry in Example 1. Compared with the control group, the addition of RAS / RAF inhibitors significantly inhibited ferroptosis.

[0051] Figure 6 This is the statistical result of the cell Lipid ROS content by flow cytometry in Example 1. Compared with the control group, the Lipid ROS content was significantly reduced after the addition of RAS / RAF inhibitors.

[0052] Figure 7 This is the result of fluorescence microscopy observation in Example 1. 50 μM 10074-G5 and 50 μM 10058-F4 were added to HT1080 cells induced by Erastin (10 μM) and RSL3 (0.2 μM). After PI staining, the addition of c-Myc inhibitor blocked ferroptosis compared with the control group.

[0053] Figure 8 This is the statistical result of cell death by flow cytometry in Example 1. Compared with the control group, the addition of c-Myc inhibitor significantly inhibited ferroptosis.

[0054] Fig. 9This is the statistical result of the cell Lipid ROS content by flow cytometry in Example 1. Compared with the control group, the Lipid ROS content was significantly reduced after the addition of c-Myc inhibitor.

[0055] Embodiment 2

[0056] RTKs / RAS / RAF / c-Myc signaling pathway inhibitors (including BMS-599626, Lapatinib, Ripretinib, Lonafarnib, AZ628, 10074-G5, and 10058-F4) block the molecular mechanism of ferroptosis.

[0057] Western blotting

[0058] Sample preparation: The protein sample concentration was determined using the protein quantification-BCA assay method. The cell protein sample and 5× loading buffer were added to a 1.5 mL centrifuge tube, mixed, and boiled in a metal bath at 100°C for 8 min to denature the protein. Electrophoresis: According to the molecular weight of the target protein, select the appropriate concentration of separation gel and concentration gel, load equal amounts of protein and protein marker into the SDS-PAGE gel wells, and run the gel at a constant voltage of 150V; Transfer: After the electrophoresis, take out the gel, cut 1 NC membrane and 6 filter papers according to the size of the gel, and spread them on the transfer instrument in the order of 3 layers of filter paper, NC membrane, gel, and 3 layers of filter paper from bottom to top, cover the transfer cover, and transfer the membrane at a constant current of 300mA; Blocking: Soak the NC membrane in 5% skim milk powder blocking solution and incubate at room temperature for 1h in a shaker; Primary antibody: Add the corresponding primary antibody to 5ml of blocking solution at a ratio of (1:1000), put in the NC membrane, incubate overnight at 4℃, and wash the membrane 3 times with TBST the next day, 10min / time; Secondary antibody: Add 5ml of blocking solution at a ratio of (1:5000) Add the corresponding secondary antibody labeled with horseradish peroxidase into TBST, put into NC membrane, incubate at 37℃ in a shaker for 50min, wash the membrane 3 times with TBST, 10min / time; imaging: take out the NC membrane, add appropriate amount of ECL (A solution: B solution = 1:1), and take pictures with a multi-color fluorescence imager.

[0059] Fig.10 The Western Blot results in Example 2 show that 50 μM 10074-G5 and 50 μM 10058-F4 were added to HT1080 cells. After 24 hours, the protein expression level of ACSL4 was detected. Compared with the control group, the c-Myc inhibitor treatment significantly reduced the expression level of ACSL4 protein.

[0060] Real-time fluorescence quantitative PCR experiment

[0061] After discarding the HT1080 cell culture medium, rinse twice with PBS. Add 1mL Trizol extract to a 1.5mL EP tube, blow for 5 minutes, add 0.2mL chloroform, shake vigorously for 10 seconds, let stand for 5 minutes, and centrifuge at 4°C, 13500rpm for 15 minutes. Take the supernatant and place it in a new EP tube, add 0.5mL isopropanol, mix gently, let stand for 10 minutes, and centrifuge at 4°C, 13500rpm for 10 minutes. Discard the supernatant, add 1mL 75% ethanol (prepared with DEPC water) to the precipitate, mix well, and centrifuge at 4°C, 10600rpm for 5 minutes. Discard the supernatant, dry at room temperature for 5-10 minutes, and dissolve the precipitate in 10μL DEPC water. After RNA extraction, use the genomic DNA removal reverse transcription kit to generate cDNA. When performing fluorescent quantitative PCR, dilute the cDNA 10 times and design the primer concentration to 10μM. Mix 5 μL 2×ChamQ Blue Universal SYBR qPCR Master Mix, 0.2 μL forward primer, 0.2 μL reverse primer, 1 μL cDNA and RNase-Free dH2O to 10 μL, add to the eight-tube strip, cover the tube tightly, centrifuge the sample briefly, place it in the fluorescence quantitative PCR instrument, set the program and collect the fluorescence signal.

[0062] Fig.11 The qPCR results in Example 2 were as follows. 50 μM 10074-G5 and 50 μM 10058-F4 were added to HT1080 cells. After 24 hours, the protein expression level of ACSL4 was detected. Compared with the control group, the c-Myc inhibitor treatment significantly reduced the mRNA expression level of ACSL4.

[0063] Fig.12 The Western Blot results in Example 2 show that 20 μM BMS599626, 20 μM Lapatinib and 20 μM Ripretinib were added to wild-type HT1080 cells, and the protein expression levels of c-Myc and ACSL4 were detected 24 hours later. Compared with the control group, the protein expression levels of c-Myc and ACSL4 were significantly downregulated after the addition of RTKs inhibitors.

[0064] Fig.13 The qPCR results in Example 2, 20 μM BMS599626, 20 μM Lapatinib and 20 μM Ripretinib were added to wild-type HT1080 cells, and the RNA expression of c-Myc and ACSL4 was detected 24 hours later. Compared with the control group, the mRNA expression levels of c-Myc and ACSL4 were significantly reduced after the addition of RTKs inhibitors.

[0065] Fig.14 The Western Blot results in Example 2 show that 10 μM Lonafarnib and 10 μM AZ628 were added to wild-type HT1080 cells, and the protein expression levels of c-Myc and ACSL4 were detected 24 hours later. Compared with the control group, the protein expression levels of c-Myc and ACSL4 were significantly downregulated after the addition of RAS / RAF inhibitors.

[0066] Fig.15 The qPCR results in Example 2 show that 10 μM Lonafarnib and 10 μM AZ628 were added to wild-type HT1080 cells, and the RNA expression of c-Myc and ACSL4 was detected 24 hours later. Compared with the control group, the mRNA expression levels of c-Myc and ACSL4 were significantly reduced after the addition of RAS / RAF inhibitors.

[0067] Based on the above experimental results, RTKs / RAS / RAF / c-Myc inhibitors can effectively block ferroptosis and inhibit ferroptosis through the molecular mechanism of downregulating c-Myc-mediated ACSL4 expression.

Claims

1. Application of RTKs / RAS / RAF / c-Myc signaling pathway inhibitors as ferroptosis inhibitors.

2. The use according to claim 1, characterized in that: The RTKs signaling pathway inhibitors include BMS-599626, Lapatinib and Ripretinib.

3. The use according to claim 1, characterized in that: The RAS / RAF signaling pathway inhibitors include Lonafarnib and AZ628.

4. The use according to claim 1, characterized in that: The c-Myc signaling pathway inhibitors include 10074-G5 and 10058-F4.

5. The use according to claim 1, characterized in that: The RTKs / RAS / RAF signaling pathway inhibitor blocks ferroptosis by downregulating c-Myc-mediated ACSL4 expression.

6. Application of RTKs / RAS / RAF / c-Myc signaling pathway inhibitors in the preparation of ferroptosis blocking drugs.

7. Application of RTKs / RAS / RAF / c-Myc signaling pathway inhibitors as ferroptosis inhibitors in the preparation of drugs for the treatment of organ damage-related diseases.

8. Application of RTKs / RAS / RAF / c-Myc signaling pathway inhibitors in inhibiting the positive regulator of ferroptosis ACSL4.

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