Application of PF-4708671 in preparation of medicine for relieving aquilaria delavayi toxin

Abstract

The invention relates to the technical field of biological medicine, and provides application of PF-4708671 in preparation of a medicine for relieving aquilaria delavayi toxin. Aiming at the problems of lack of specific therapeutic drugs, difficulty in reversing cell injury, difficulty in controlling systematic toxicity and lack of intervention strategies for signal pathways in the field of PLTX treatment, the invention proposes that a small-molecule inhibitor PF-4708671 is used for reversing cell injury caused by PLTX for the first time on the basis of further exploring a toxic action mechanism of PLTX. It is found through experiments that the PF-4708671 can remarkably relieve related damage of cellular morphology, cytoskeleton, multiplication capacity, migration and invasion capacity and adhesion capacity caused after PLTX acts on HaCaT cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and relates to the application of S6K1 as a therapeutic target for palytoxin, specifically to the application of PF-4708671 in the preparation of palytoxin-relieving drugs, and further to the application of the S6K1 inhibitor PF-4708671 in reversing cell damage caused by PLTX. Background Art

[0002] Palytoxin (PLTX) is a potent marine biotoxin. The clinical manifestations of PLTX poisoning include dyspnea, arrhythmia, hemolysis, muscle pain, renal failure, etc., and in severe cases, it can lead to multiple organ failure and death. At present, there is a lack of specific drugs for the treatment of PLTX poisoning. The existing treatment strategies mainly include supportive treatment and antidotes. Although some experimental treatments such as gene therapy and nanotechnology have shown certain potential, further research is still needed. Preventing exposure is the most effective strategy. Further advancing the rescue of PLTX poisoning requires understanding the mechanism of action of PLTX and targeted drug treatment. However, its mechanism of action is complex and its toxicity is extremely strong, making it difficult to develop targeted antidotes. Existing studies have shown that PLTX disrupts the cell ion balance by interfering with the Na+ / K+-ATPase, triggering a series of cell damages such as calcium ion overload, cell swelling, and membrane potential collapse, ultimately leading to cell death and systemic toxic reactions, and even endangering life. For this basic theory, existing antidotes include antioxidants, calcium channel blockers, etc., but there is still a lack of intervention strategies for signal pathways. At present, the specific molecular mechanism of PLTX-induced cell damage is still unclear, and there are still gaps in the research in this field. Against this background, further studying the specific molecular mechanism of PLTX action and developing more effective therapeutic drugs have important clinical significance.

[0003] The p70 ribosome S6 kinase 1 (S6K1) is a key kinase in the mTOR signaling pathway and is regulated by mTORC1. After activation, S6K1 mainly regulates protein synthesis, apoptosis, and proliferation by affecting the activities of downstream molecules, and also affects the cytoskeleton. The mTORC1 pathway in which it is located is related to various diseases such as cancer, type II diabetes, and obesity, and can regulate neuronal signals. Among the downstream molecules of S6K1, GSK3β plays a certain role in processes such as cholinergic metabolism, axonal transport, synaptic effects, inflammation, and the cell cycle, and its overexpression plays an inducing role in various cell apoptoses; the expression level of β-catenin is related to inflammatory responses, cell proliferation, stem cell renewal and differentiation, tissue development and homeostasis, and the occurrence and development of cancer; BAD promotes cell survival and inhibits apoptosis; the excessive accumulation of tau leads to the decomposition of microtubules, destroys the cytoskeleton, and affects the phenotype of microglia; E-cadherin is an important adhesion molecule, and its downregulation is an important mechanism for the occurrence of epithelial-mesenchymal transition (EMT). The regulation of the above processes can be achieved through the mTOR / S6K1 pathway. Most literature shows that the activation of S6K1 will lead to stronger cell proliferation, migration, and invasion abilities, however, this is different from the phenotypic changes we actually observed in HaCaT cells. However, there are also literature indicating that the phosphorylation modification of PDK1 by S6K1 can inhibit the activity and carcinogenic function of the AKT kinase. In summary, the phenotypic changes caused by the alteration of the expression and phosphorylation levels of the downstream molecules of S6K1, including the above-mentioned molecules, are closely related to the cell phenotypic changes caused by PLTX.

[0004] PF-4708671 is a cell-permeable and highly specific inhibitor of S6K1 with an IC 50 value of 160 nM. It is the first discovered inhibitor of S6K1 and can affect cell proliferation, survival, and metabolism by regulating the mTORC1 / S6K1 signaling pathway. PF-4708671 has a more obvious inhibitory effect on S6K1 than on other related kinases, such as RSK2 and MSK1. In addition, PF-4708671 can prevent S6K1 from phosphorylating its substrates, including ribosomal S6 protein, mTOR, and Rictor. PF-4708671 can also enhance the phosphorylation of Thr229 and Thr389 sites in S6K1, resulting in an increase in S6K1 activity, but the increase induced by PF-4708671 is relatively weak compared to the increase in S6K1 activity stimulated by IGF1. S6K1 is involved in the formation of stress granules (SGs) in mild oxidative stress, resulting in the inhibition of protein translation. PF-4708671 inhibits S6K1-mediated S6 phosphorylation, which can reduce the formation of SGs in mild oxidative stress and promote the cell translation process.

[0005] The role of S6K1 in the damage of PLTX-infected cells and the alleviating effect of PF-4708671 on PLTX toxin have not been reported. Summary of the Invention

[0006] The present invention aims at the treatment problem of PLTX toxin alleviation. The purpose is to provide a new medical use of S6K1 inhibitors, and also to provide the application of S6K1 as a target in alleviating the damage of PLTX to cells. Specifically, it provides the application of the S6K1 inhibitor PF-4708671 in the preparation of drugs for alleviating PLTX.

[0007] In view of the problems in the field of PLTX treatment, such as the lack of specific therapeutic drugs, the irreversibility of cell damage, the difficulty in controlling systemic toxicity, and the lack of intervention strategies for signal pathways, on the basis of further exploring the mechanism of action of PLTX, the present invention first proposes to use the small molecule inhibitor PF-4708671 to reverse the cell damage caused by PLTX. The present invention discovers through experiments that PF-4708671 can significantly reduce the related damage of cell morphology, cytoskeleton, proliferation ability, migration and invasion ability, and adhesion ability caused by PLTX acting on HaCaT cells.

[0008] Based on the above research, the specific technical solutions of the present invention are as follows:

[0009] In the first aspect of the present invention, there is provided the application of an S6K1 inhibitor in the preparation of a drug for alleviating PLTX.

[0010] Preferably, the S6K1 inhibitor is selected from the small molecule inhibitor PF-4708671.

[0011] The results of cell function experiments show that the inhibitor PF-4708671 plays a role in reversing and repairing the related damage of cell morphology, cytoskeleton, proliferation ability, migration and invasion ability, and adhesion ability caused by PLTX acting on cells.

[0012] Therefore, the drug of the present invention is essentially a drug for reversing cell damage, and further a drug for affecting the expression and phosphorylation level of downstream molecules regulated by S6K1.

[0013] The drug for alleviating PLTX described in the present invention is PF-4708671 or its derivative as the sole active ingredient or a pharmaceutical composition containing PF-4708671 or its derivative.

[0014] The drug or pharmaceutical composition of the present invention can be formulated into any dosage form with pharmaceutically common excipients. For example, it can be an oral preparation (such as tablets, capsules, granules, decoctions, oral ampoules, dripping pills, etc.), an injection preparation (such as an intravenous injection preparation), a transdermal absorbent (such as an iontophoretic agent, a patch plaster, an aerosol, a film, a sponge, etc.).

[0015] In a second aspect of the present invention, there is provided a pharmaceutical composition for relieving PLTX, which is composed of PF-4708671 or its derivatives and pharmaceutically acceptable excipients.

[0016] PF-4708671 or its derivatives of the present invention are used for relieving the stings of Anemonia sulcata, and are particularly suitable for groups with a high probability of being stung by Anemonia sulcata, such as offshore workers, coastal residents, travelers, etc. PF-4708671 or its derivatives of the present invention can be particularly used for preventing the stings of Anemonia sulcata, that is, administering the drug in advance to people who may encounter the stings of Anemonia sulcata, and the administration methods are not limited to oral administration, injection, etc.

[0017] Furthermore, the pharmaceutical composition of the present invention can be used in combination with other pharmaceutical compositions for relieving PLTX.

[0018] Functions and effects of the invention

[0019] 1) The present invention provides a small molecule inhibitor PF-4708671, which can inhibit the activity of S6K1 in cells, thereby affecting the expression and phosphorylation levels of its downstream molecules, and playing a role in reversing and repairing the related damages of cell morphology, cytoskeleton, proliferation ability, migration and invasion ability, and adhesion ability caused by the action of PLTX on cells. The above effects have been experimentally verified in the examples of the present invention, expanding the clinical application scope of PF-4708671. At the same time, using small molecule inhibitors to treat PLTX poisoning has the advantages of easy large-scale production, low cost, and convenient access. This drug is expected to provide feasibility and theoretical basis for the clinical treatment of PLTX poisoning, promote the research progress in the field of PLTX treatment, and provide new ideas for the treatment of poisoning by PLTX and similar toxins.

[0020] 2) During the research process of the present invention, it involves two major parts: multi-omics combined analysis and cell-level experiments. Using PLTX-sensitive cells, namely HaCaT cells, for research, comprehensively and convincingly verifying the effect of the inhibitor PF-4708671. The research methods used are comprehensive and innovative, providing ideas for related mechanism research.

[0021] 3) PF-4708671 used in the present invention is an S6K1 inhibitor. A mechanism route of PLTX action involving the key molecule S6K1 and related to the expression and phosphorylation of downstream molecules of S6K1 was initially constructed, which promoted the research on the specific mechanism of PLTX action, filled the research gap to a certain extent, and is expected to provide a theoretical basis for the clinical research of PLTX. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Shows the experimental flow chart of the present invention;

[0023] Figure 2 Shows that in the examples of the present invention, multi-omics analysis found that S6K1 is the key kinase for PLTX-induced cell damage. Among them, A shows the results of GO enrichment analysis of differentially expressed genes screened by transcriptome sequencing before and after PLTX treatment; B shows the results of domain prediction of differentially expressed proteins screened by proteome sequencing before and after PLTX treatment; C shows the results of kinase substrate enrichment analysis (left) and the corresponding changes in kinase protein expression levels (right); D shows the results of GO enrichment analysis of proteins with unchanged expression levels but significant changes in phosphorylation levels.

[0024] Figure 3 Shows that in the examples of the present invention, CCK8 assay shows that the application of PF-4708671 can reverse the damage of PLTX to cell proliferation ability.

[0025] Figure 4 Shows that in the examples of the present invention, cell scratch assay shows that after the application of PF-4708671, the damage of PLTX to cell migration and repair ability is reversed.

[0026] Figure 5 Shows that in the examples of the present invention, transwell assay shows that the application of PF-4708671 can reverse the damage of PLTX to cell migration ability.

[0027] Figure 6 Shows that in the examples of the present invention, transwell assay shows that the application of PF-4708671 can reverse the damage of PLTX to cell invasion ability.

[0028] Figure 7 Shows that in the examples of the present invention, CCK8 assay shows that the application of PF-4708671 can reverse the damage of PLTX to cell adhesion ability. DETAILED DESCRIPTION OF THE INVENTION

[0029] The following examples and experimental examples further illustrate the present invention and should not be construed as limiting the present invention. The examples do not include a detailed description of traditional methods, such methods being well known to those of ordinary skill in the art and having been described in many publications.

[0030] Unless otherwise defined, all professional and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention, and the preferred methods and materials described in the specific embodiments are for illustrative purposes only.

[0031] I. Experimental methods adopted in the examples

[0032] Figure 1 The experimental process of the present invention is shown as follows:

[0033] 1. Multi-omics analysis

[0034] The original data of transcriptome sequencing was quality controlled by fastp, then the data was aligned to the hg38 reference genome using hisat2, and then quantification was completed using featureCounts. DESeq2 was used for differential gene screening, and the R package clusterProfiler was used for enrichment analysis; ProteomeDiscoverer 1.4 was used for the identification and quantification of phosphorylated proteome and proteome data. DEqMS was used for differential expression gene screening, interproscan was used for the prediction of differentially expressed protein domains, t-test was used for the screening of differentially phosphorylated peptides, and the R package KSEAapp was used for kinase substrate enrichment analysis.

[0035] 2. CCK8-cytotoxicity experiment

[0036] HaCaT cells were cultured routinely. Cells in good growth state and in the logarithmic growth phase were selected for digestion, pipetted and mixed into a single cell suspension, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in DMEM complete medium containing 10% FBS and quantified to 1×10 5 / mL for standby. Take a 96-well plate, set up 3 duplicate wells and 1 blank control well for each experimental group, add PBS to the outermost well to prevent the evaporation effect during cell culture from affecting cell growth, add 100μL / well of cell suspension as planned, and after the cell plating is completed, place the 96-well plate in a cell culture incubator for 24h; after the cells are completely attached to the wall, continue to add 100 μL of drug solution prepared with complete culture medium to each well according to the intervention plan, and continue to culture in the incubator for 24h. Then add 10 μL of CCK8 solution to each well, incubate the culture plate in the incubator for about 30min, use an enzyme reader to measure the OD value at 450nm and calculate the cell survival rate.

[0037] 3. Cell scratch assay

[0038] HaCaT cells were routinely cultured, and cells in good growth state and in the logarithmic growth phase were selected for digestion. The cells were mixed by pipetting to form a single cell suspension, centrifuged at 800 rpm for 5 min, the supernatant was discarded, and DMEM complete medium containing 10% FBS was added to resuspend the cells and count them. A horizontal line was marked on the bottom of a 6-well plate with a marker pen, and then about 10 cells were inoculated into each well. 6 cells were placed in a cell culture incubator for 24 h. When the cell confluence reached about 80%, the 6-well plate was taken out and a 200 μL pipette tip was used to perpendicularly hold the well surface to scratch in a direction perpendicular to the horizontal line marked on the bottom surface. After the scratching was completed, the old culture medium was aspirated and the cells were washed with PBS to remove the scratched cells so that the scratches were clearly visible. According to the intervention plan, 200 μL of drug solution prepared with low-serum DMEM culture medium was added to each well to intervene in the cells. The cells were placed in a cell culture incubator for culture. The 6-well plate was taken out at 0, 6 h, 12 h, 24 h, and 48 h, and observed and photographed under a microscope. The results were quantified using ImageJ 1.53 software.

[0039] 4. Transwell-migration assay

[0040] HaCaT cells were routinely cultured, and cells in good growth state and in the logarithmic growth phase were selected for digestion. The cells were mixed by pipetting to form a single cell suspension, centrifuged at 800 rpm for 5 min, the supernatant was discarded, serum-free DMEM medium was added to resuspend the cells and counted. The quantitative value of the chamber migration experiment was 2×10 6cells / mL; Take out the 24-well plate and the chamber. Pre-add 600 μL / well of DMEM medium containing 20% FBS to the 24-well plate. Gently place the chamber into the 24-well plate with forceps, avoiding the formation of air bubbles between the bottom of the chamber and the medium. Take 50 μL of the cell suspension for intervention as planned, mix well and inoculate it into the chamber, with a total volume of 100 μL / chamber, and then place it in a cell incubator for 24 h; Take out the 24-well plate, aspirate the old medium, and gently wipe the cells in the upper chamber with a cotton swab; Add 600 μL of 4% paraformaldehyde to the 24-well plate, place the chamber in and fix for 15 min; Aspirate and discard the fixing solution, and wash the chamber once with PBS; Add 600 μL of 0.1% crystal violet solution to the 24-well plate, place the chamber in and stain for 10 min; Take out the chamber, wash it 3 times with PBS, air dry appropriately, then observe and take pictures under a microscope, and quantify the results with ImageJ 1.53 software.

[0041] 5. Transwell-Invasion Assay

[0042] The method of the Transwell chamber invasion assay is similar to the above migration assay, mainly adding the step of coating with Matrigel. That is, take out the Matrigel the night before the experiment, place it at 4°C to thaw slowly, and at the same time place the required pipette tips, etc. at -20°C for pre-cooling. The next day, place the 24-well plate on ice, take out the Matrigel, dilute it according to the ratio of Matrigel: serum-free DMEM medium = 1:8, and coat 60 μL per chamber, then place the 24-well plate in the incubator for 2 h; Aspirate the excess Matrigel in the chamber, add 100 μL of serum-free DMEM medium, and place it in the incubator for 30 min for basement membrane hydration; The cell suspension inoculated in the chamber invasion assay is quantified to 2×10 7 cells / mL; The subsequent method steps are the same as those of the chamber migration.

[0043] 6. CCK8 Cell Adhesion Assay

[0044] (1)Coating and Blocking of the Culture Plate

[0045] Add 600 μL / well of 10 μg / mL fibronectin (human fibronectin) to the 96-well plate and coat at room temperature for 1 h. After recovering the coating solution, add 200 μL / well of 1% fetal bovine serum and incubate in a constant temperature incubator at 37°C for 1 h for blocking. Then carefully aspirate the fetal bovine serum and add serum-free DMEM medium to wash the well plate 2 times.

[0046] (2)Cell Inoculation and Treatment

[0047] Cultivate HaCaT cells routinely. Select cells with good growth status and in the logarithmic growth phase for digestion. Pipette and mix well to form a single-cell suspension. Centrifuge at 800 rpm for 5 min, discard the supernatant, add DMEM complete medium containing 10% FBS to resuspend the cells and count them, and quantify to 3×10 5 cells / mL; Take the coated 96-well plate, set 3 replicate wells and 1 blank control well for each experimental group. Add PBS to the outermost wells to prevent the evaporation effect during cell culture from affecting cell growth. Add 100 μL of cell suspension per well in sequence. After plating, place it in the cell culture incubator for 24 h; After the cells adhere, aspirate the original medium. According to the intervention plan, add 100 μL of drug solution prepared with low-serum medium to each well. Place the 96-well plate in the cell culture incubator and continue to culture for 24 h;

[0048] Determine the absorbance by CCK8 method. Aspirate the original medium, wash 1-2 times with PBS, add 110 μL / well of CCK-8 dilution solution diluted at a ratio of CCK-8: serum-free double antibody medium = 1:10, incubate at 37 °C for 30 min, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at 450 nm and calculate the adhesion value. Specific embodiments

[0049] Example 1 Omics analysis to lock the key kinase S6K1 causing cell damage by PLTX

[0050] To comprehensively understand the key role of S6K1 in PLTX-induced HaCaT cell damage and further verify the key role of the S6K1 inhibitor (PF-4708671) in reversing cell damage, the present invention performed transcriptome and proteome analyses on HaCaT cells before and after PLTX treatment and found that differentially expressed genes were mostly enriched in functions related to protein phosphorylation ( Figure 2 A, B), and it was speculated that the change in kinase expression level might be related to the toxic effect of PLTX. Then, kinase substrate enrichment analysis was combined with phosphoproteomic data. Among them, the expression level of RPS6KB1 encoding S6K1 was significantly increased and the kinase activity was predicted to be activated. Therefore, S6K1 might be the key kinase causing cell damage by PLTX ( Figure 2 C). The proteomic data was correlated with the phosphoproteomic data, and GO enrichment analysis was performed on genes with no change at the protein level but significant change at the phosphorylation level. It was found that these genes were mainly enriched in cell components such as adhesion proteins and cytoskeletons ( Figure 2 D).

[0051] The above results indicate that PLTX may regulate the phosphorylation processes of proteins such as cytoskeleton and cell adhesion junction proteins by upregulating the expression of S6K1, leading to cell damage; PF-4708671, as an S6K1 inhibitor, has the potential to inhibit cell damage caused by PLTX by inhibiting the expression of S6K1.

[0052] Example 2 Verification of cell damage repair

[0053] To verify the role of PLTX in cell damage and the role of PF-4708671 inhibitor in cell damage repair, the target cells selected were HaCaT cells with high sensitivity to PLTX. First, resuscitate the cells: Take out the cryopreservation tube from -80 °C, and let it melt as soon as possible in a 37 °C metal bath for 1 min; Take out the cryopreservation tube from the 37 °C metal bath, add 1 mL of complete DMEM medium, and mix well; Centrifuge at 800 rpm for 5 min, discard the supernatant, add DMEM medium containing 10% FBS to resuspend the cells, and evenly inoculate them in a 60 mm culture dish, and incubate statically in a 5% CO2, 37 °C incubator; Replace the culture medium once the next day and continue culturing; When the cell density reaches 80-90%, use trypsin to digest the cells, determine the number of wells to be plated according to subsequent experiments, and continue culturing; And perform corresponding PLTX and / or PF-4708671 inhibitor interventions on the cells. The cell intervention protocol designed in the present invention is: control (control group, no intervention), PLTX IC 10 (1.5×10 -12 μg / mL), PLTX IC 20 (7.8×10 -9 μg / mL), PLTX IC 30 (7.2×10 -8 μg / mL), PF-4708671, PLTX IC 10 +PF-4708671, PLTX IC 20 +PF-4708671, PLTX IC 30 +PF-4708671.

[0054] Example 3 PF-4708671 promotes the reverse repair of damaged cells

[0055] To comprehensively understand the role of PF-4708671 inhibitor in repairing PLTX-induced cell damage, the present invention found through CCK8-cytotoxicity test ( Figure 3 ) and cell function experiments (cell scratch experiment, Transwell-migration, Transwell-invasion experiment and CCK8-adhesion experiment) that after applying PF-4708671, the damage of PLTX to the proliferation, migration, invasion and adhesion abilities of HaCaT cells was significantly reversed (Figures 4 - 7 )。

[0056] The above results indicate that treatment with the small molecule inhibitor PF-4708671 can inhibit the activation of S6K1 caused by PLTX, and may further affect the expression and phosphorylation levels of downstream molecules of S6K1, thereby reversing the various damaging effects of PLTX on cell proliferation, migration, invasion, and adhesion. Therefore, the small molecule inhibitor PF-4708671 will be an effective method for treating the toxic damage caused by PLTX in the future. Especially for the damage mediated by S6K1, it has good clinical treatment prospects.

[0057] The parts not described in this invention are the same as the prior art or are implemented using the prior art. The applicant declares that this invention uses the above embodiments to illustrate the detailed method of this invention, but this invention is not limited to the above detailed method, that is, it does not mean that this invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvement to this invention, the equivalent substitution of each raw material of the products of this invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and the disclosure scope of this invention.

Claims

1. Use of an S6K1 inhibitor in the preparation of a drug for relieving palytoxin (PLTX).

2. The application according to claim 1, characterized in that, The S6K1 inhibitor is selected from the small molecule inhibitor PF-4708671.

3. The application according to claim 1 or 2, characterized in that, The drug is a drug for reversing cell damage caused by PLTX.

4. The application according to claim 3, wherein The drug for reversing cell damage is a drug that exerts a reversing and repairing effect on the relevant damage of cell morphology, cytoskeleton, proliferation ability, migration and invasion ability, and adhesion ability caused by the action of PLTX on cells.

5. The application according to claim 4, characterized in that The drug is a drug that affects the expression and phosphorylation level of downstream molecules regulated by S6K1.

6. The application according to claim 1 or 2, characterized in that, The drug for relieving PLTX is a drug composition taking PF-4708671 as the sole active ingredient or containing PF-4708671.

7. The application according to claim 1 or 2, characterized in that, The drug is selected from oral preparations, injection preparations or transdermal absorbents.

8. A pharmaceutical composition for relieving PLTX, characterized in that, It is composed of PF-4708671 and pharmaceutically acceptable excipients.

9. The pharmaceutical composition for alleviating PLTX according to claim 8, characterized in that, This drug composition is used in combination with other drug compositions for relieving PLTX.

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