Application of S100A8 as a therapeutic target for vascular malformations

Abstract

This invention belongs to the field of disease treatment technology and discloses the application of S100A8 as a therapeutic target for vascular malformations. This invention validates the therapeutic effect of montelukast (MS) on DDX24-deficient vascular malformations in vitro and in vivo. Immunoprecipitation results show that MS stabilizes DDX24 protein expression by inhibiting ubiquitination in the cytoplasm. Mass spectrometry identifies S100 calcium-binding protein A8 (S100A8) as the direct binding target of MS in endothelial cells (ECs), and this is verified by cell thermal migration assays, drug affinity target stability, molecular docking simulations, and surface plasmon resonance analysis. Through direct interaction with S100A8, MS promotes the formation of a complex between S100A8 and the DDX24-Q motif. Our study reveals a novel function of MS in treating vascular malformations through the novel target S100A8, providing a new strategy for the treatment of this disease.

Description

Technical Field

[0001] This invention belongs to the field of disease treatment technology, and more specifically, relates to the application of S100A8 as a therapeutic target for vascular malformations. Background Technology

[0002] Montelukast sodium (MS) has long been considered a classic anti-inflammatory drug for treating asthma. MS exerts its effect by targeting the cysteine ​​leukotriene receptor (CysLTR1), which binds to leukotriene D4 (LTD4) and E4 (LTE4) to mediate inflammatory signaling. In addition to asthma and multiple sclerosis, MS is also used for various cardiovascular diseases, such as atherosclerosis, myocardial infarction, and cardiac fibrosis.

[0003] Vascular malformations (VMs) are abnormalities in blood vessel formation that can occur in various types of vessels, such as arteries, veins, lymphatic vessels, or capillaries, significantly impacting patients' quality of life and mortality. In most cases, VMs are caused by abnormal vascular development, leading to altered angiogenesis. A novel type of VM, called multiple organ venous and lymphatic defects syndrome (MOVLD syndrome), has been previously reported, with symptoms primarily including stenosis or occlusion of the portal and hepatic veins, accompanied by the formation of collateral circulation. Currently, there is no effective treatment for MOVLD syndrome.

[0004] Chinese patent application number 201910705697.5 discloses compounds for treating vascular malformations. The paper points out that CysLTR1 antagonists, such as montelukast, can upregulate expression in vascular smooth muscle cells (DDX24), exhibiting a certain therapeutic or ameliorative effect on vascular malformations. Interestingly, increasing evidence suggests that CysLT1R is not the only cellular target regulated by MS. For example, the bile acid receptor GPBAR1, a novel ligand for MS in macrophages, can alleviate inflammation in colitis. This indicates that, besides CysLT1R, potential targets and mechanisms of MS remain to be explored. Summary of the Invention

[0005] In order to overcome the shortcomings of the prior art, the purpose of this invention is to provide the application of S100A8 as a therapeutic target for vascular malformations.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] The application of S100A8 as a binder for montelukast in the treatment of vascular malformations caused by DDX24 knockdown.

[0008] This invention demonstrates that MS (montelukastner) binds directly to the binding pocket of S100A8 with moderate affinity (Kd = 1.07 μM). S100A8 belongs to the largest subgroup in the Ca2+-binding EF-hand superfamily and is essential for calcium ion influx influencing endothelial cell migration and proliferation. Therefore, S100A8 can be identified as a novel MS binder that can effectively attenuate DDX24 deficiency-induced angiogenesis.

[0009] Preferably, in the above applications, S100A8 combined with montelukast enhances the therapeutic effect of montelukast on vascular malformations caused by DDX24 knockdown.

[0010] This invention, based on the premise that S100A8 is a therapeutic target for treating vascular malformations caused by MS, investigated the therapeutic effect of overexpressing S100A8 on MS. The results showed that MS could inhibit the increase in endothelial migration induced by DDX24 deficiency, but overexpression of S100A8 could reverse the increase in endothelial migration caused by DDX24 knockdown; furthermore, MS could inhibit the increase in endothelial migration induced by DDX24 overexpression.

[0011] Meanwhile, MS can inhibit the increase in endothelial tube formation induced by DDX24 deficiency; overexpression of S100A8 can reverse the increase in endothelial tube formation caused by DDX24 knockdown; and overexpression of S100A8 can inhibit the increase in endothelial tube formation caused by DDX24 deficiency.

[0012] In conclusion, it can be determined that S100A8 can enhance the efficacy of MS treatment.

[0013] Therefore, this invention also protects the use of substances that promote S100A8 expression in the preparation of functional products for treating vascular malformations caused by DDX24 knockdown with montelukast.

[0014] Preferably, the functional product serves as an enhancer for montelukast in treating vascular malformations caused by DDX24 knockdown.

[0015] The present invention also protects the use of substances that promote S100A8 expression and montelukast as pharmaceutical compositions in the preparation of medicaments for treating vascular malformations caused by DDX24 knockdown.

[0016] Preferably, the substance that promotes S100A8 expression includes any one of the following:

[0017] (i) Targeting S100A8 or the S100A8 transcript as the target sequence and being able to enhance the expression of the S100A8 gene expression product;

[0018] (ii) A construct containing S100A8, or a complementary sequence of S100A8, and capable of forming an enhanced S100A8 gene expression product after being transfected into the body;

[0019] (iii) Cells or constructs that overexpress the S100A8 gene sequence.

[0020] More preferably, the substances that promote S100A8 expression are plasmids, viruses, and gene-edited cells that overexpress S100A8.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] This invention validates the therapeutic effect of MS on DDX24-deficient vascular malformations through in vitro and in vivo experiments. Immunoprecipitation results showed that MS stabilizes DDX24 protein expression by inhibiting ubiquitination in the cytoplasm. Mass spectrometry identified S100 calcium-binding protein A8 (S100A8) as the direct binding target of MS in endothelial cells (ECs), and this was verified by cell thermal migration assay (CETSA), drug affinity target stability (DARTS), molecular docking simulation, and surface plasmon resonance (SPR) analysis. Through direct interaction with S100A8, MS promotes the formation of a complex between S100A8 and the DDX24-Q motif. Our study reveals a novel function of MS in treating vascular malformations through the novel target S100A8, providing a new therapeutic option for the treatment of vascular malformations. Attached Figure Description

[0023] Figure 1 The results showed that montelukast (MS) could alleviate DDX24 deficiency-induced vascular malformations; Figure 1 A and Figure 1 B is a representative image (A) and quantification (B) of vascular endothelial cell migration after MS inhibition of DDX24 knockdown, scale bar 200 μm; Figure 1 C and Figure 1 D represents the representative image (C) and quantification (D) of vascular endothelial cell tube formation after MS inhibition of DDX24 knockdown, with a scale bar of 200 μm; Figure 1 EG represents the ISV overbranching in zebrafish embryos after MS-injection of ddx24 morpholino (E), the incidence of abnormal ISVs (F), and the number of abnormal ISVs (G). White rectangles represent magnified views of local regions, with a scale bar of 10 μm. Figure 1 H represents the protein level of DDX24 in HUVECs transfected with DDX24 siRNA or control and treated with MS (10 μM) for 12 hours using Western blot. Figure 1I is a Western blot showing the Ddx24 protein level in zebrafish embryos injected with ddx24 morpholino and treated with MS (10 μM) for 48 hours; Figure 1 J and 1K are NSGs treated with MS after DDX24 knockdown. TM Representative images of Matrigel (J) and blood vessel (CD31) staining in mice, magnified views of local areas indicated by white boxes, scale bar 10 μm, n=4; statistical analysis was performed using one-way ANOVA. Figure 1 B, 1D, 1F, 1G, 1I, 1K), Student's t-test ( Figure 1 H); All data are expressed as mean ± SEM; ns, no significant difference, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001;

[0024] Figure 2 The results show that MS has other targets besides CysLTR1; Figure 2 A shows the protein level of CysLTR1 in HUVECs transfected with CysLTR1 siRNA or in control HUVECs as displayed by Western blot. Figure 2 B shows representative images and quantifications of HUVECs transfected with CysLTR1 siRNA or controls detected by Transwell assay, with a scale bar of 200 μm. Figure 2 C represents representative images and quantifications of HUVECs transfected with CysLTR1 siRNA or controls in tube formation assays, with a scale bar of 200 μm; statistical analysis was performed using one-way ANOVA; all data are expressed as mean ± SEM; *P<0.05, **P<0.01;

[0025] Figure 3 This indicates that S100A8 is the direct target of MS; Figure 3 A represents the chemical structure of biotin-conjugated montelukast; Figure 3 B is a flowchart of target selection; Figure 3 C represents the mass spectrometry analysis of global proteins in HUVECs and the identification of the S100A8 peptide; Figure 3 D represents the expression of S100A8 protein in HUVECs treated with MS (10 μM) or control (DMSO) at a specified temperature using Western blot analysis (detected by CETSA). Figure 3 E represents the expression of S100A8 protein detected by Western blot after HUVECs were treated with a specified dose of MS for 1 hour before and after digestion with 1 μg / mL enzyme E. Figure 3 F represents the direct interaction between MS and S100A8 as shown by SPR analysis; Figure 3G is a schematic diagram of S100A8 and its mutants. The S100A8 mutant contains the deleted helical domain 1(ΔH1)H:helix structure; Figure 3 H represents the molecular simulation of the MS binding site S100A8; Figure 3 I represents the SPR analysis combining MS and S100A8-MUT;

[0026] Figure 4 The purification and affinity of S100A8 and DDX24 proteins with MS are shown. Figure 4 A represents the S100A8 protein purified from E. coli. SDS-PAGE (15% gel) was performed on S100A8-6xHIS using a Ni-NTA column. The red rectangle indicates the location of the fusion protein S100A8-6xHIS (12.7 kDa). Figure 4 The left image (B) shows the chromatogram of purified DDX24 protein through a Superdex 7516 / 600 gel filtration column; the right image shows the SDS-PAGE analysis of DDX24 by Ni-NTA size exclusion chromatography, with the red rectangles indicating the positions of the DDX24 protein. Figure 4 C represents the SPR analysis combining MS and DDX24;

[0027] Figure 5 This demonstrates the validation of S100A8 knockdown in endothelial cells; Figure 5 A shows the protein level of S100A8 in HUVECs transfected with S100A8 siRNA or in control HUVECs as displayed by Western blot. Figure 5 B represents the expression of S100A8 mRNA transfected with S100A8 siRNA or control mRNA by RT-qPCR; statistical analysis was performed using one-way ANOVA; data are expressed as mean ± SEM; ns, no significant difference, *P<0.05 and ***P<0.001;

[0028] Figure 6 The results showed that MS combined with S100A8 inhibited angiogenesis; Figure 6 A and Figure 6 B shows representative images (A) and quantification (B) of HUVECs transfected with S100A8 siRNA or control, processed by Transwell assay, with a scale bar of 200 μm. Figure 6 C and 6D are representative images (C) and quantification (D) of HUVECs detected by tube formation assay with S100A8 siRNA or control, with a scale bar of 200 μm; Figure 6 E represents the expression of DDX24 in HUVECs transfected with control or S100A8 siRNA, MS (10 μM) treatment or no treatment for 12 hours by Western blot. Figure 6F is a fluorescence staining image showing the expression of DDX24 in HUVECs treated with MS (10 μM) and without MS for 12 hours. Scale bar: 10 μm. Figure 6 G represents DDX24 ubiquitination as shown by Western blot. After transfection of HEK293T cells with S100A8 siRNA or control, HA-ubiquitin and Flag-DDX24 were overexpressed, followed by treatment with MG132 (2 μM) and / or MS (10 μM) for 12 hours. Figure 6 H and 6I are representative images (H) and quantification (I) of HUVECs overexpressing S100A8 after transfection with S100A8 siRNA, detected by Transwell assay, with a scale bar of 200 μm; Figure 6 J and 6K are representative images (J) and quantification (K) of HUVECs transfected with S100A8 siRNA and overexpressing S100A8, as detected in tube formation experiments. Scale bar: 200 μm. Statistical analysis was performed using Student's t-test. Figure 6 B and 6D), one-way ANOVA (6I and 6K); all data are expressed as mean ± SEM; ns, no significance, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001;

[0029] Figure 7 The MS signal facilitates the interaction between S100A8 and DDX24. Figure 7 A shows the interaction between S100A8 and exogenous Flag-DDX24 in HEK293T cells as detected by Western blot. Figure 7 B represents the co-IP analysis of the interaction between S100A8 and Flag-DDX24 in HUVECs before and after MS treatment; Figure 7 C represents a representative image and white arrow diagram of the co-location of S100A8 and DDX24 in HUVECs, with a scale bar of 10 μm; Figure 7 D is a schematic diagram of the full-length and truncated DDX24 transcripts; Figure 7 E is a Western blot image of HEK293T cells. After treatment with MS (10 μM) for 12 hours, co-IP detection showed that Myc-S100A8 interacted with a specified truncation of Flag-DDX24. Figure 7 F and 7G are representative images showing the colocalization of endogenous S100A8 with transfected Flag-DDX24-N2 (F) or -C2 (G) in HUVECs by immunostaining. The inset on the right shows the colocalization coefficients, with a scale bar of 10 μm.

[0030] Figure 8 This demonstrates the interaction between MS-enhanced exogenous DDX24 and S100A8; Figure 8 A represents the immunoblotting analysis of the interaction between exogenous Myc-S100A8 and Flag-DDX24 in HEK293T cells transfected with Flag-DDX24 and Myc-S100A8. Figure 8 B represents the co-location of exogenous Flag-DDX24 and Myc-S100A8 in HUVECs; Figure 8 C is a representative image of co-localization of DDX24-FL and S100A8 in endothelial cells. The right image shows the co-localization coefficients, with a scale bar of 10 μm.

[0031] Figure 9 S100A8 shows that it can enhance the treatment of MS; Figure 9 A represents the images and quantification of HUVECs transfected with S100A8 siRNA or control and then overexpressed with S100A8-Myc under MS by Transwell assay, scale bar 200 μm; Figure 9 B shows the images and quantification of HUVECs after treatment with siS100A8 or control followed by overexpression of S100A8-Myc in the tube formation experiment, under MS. Scale bar, 200 μm. Statistical analysis was performed using one-way ANOVA. All data are expressed as mean ± SEM. ns, no significant difference, *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Detailed Implementation

[0032] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific drawings and embodiments. Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used are commercially available unless otherwise specified.

[0033] Cell Culture and Cell Lines: Human umbilical vein endothelial cells (HUVECs) were cultured in ECM supplemented with 10% fetal bovine serum. HUVECs were used for experiments up to passage 10. The medium was changed every other day and separated from TrypLESelect cells for passage upon reaching confluence. Human embryonic kidney 293T (HEK293T) cells were cultured in DMEM containing 10% fetal bovine serum, 1% penicillin, and streptomycin. The cells were cultured at 37°C in 5% CO2. Cells were identified by short tandem repeat (STR) analysis and confirmed to be free of mycoplasma contamination before use.

[0034] Animal experiments were approved by the Ethics Guidelines for Animal Experimentation Institutions (#00498) formulated by the Fifth Affiliated Hospital of Sun Yat-sen University.

[0035] Zebrafish Experiment: Embryos were collected by mating zebrafish and stored in E3 medium at 28.5°C. For zebrafish models lacking ddx24, 1.5 ng of ddx24 MO (5'-TAATCTCTGTGCCCCTACCGTCTTC-3') was injected into the yolk sac in embryos at stages 1–4. Healthy embryos were selected into 6-well plates at 6 hours post-fertilization (hpf) and exposed to different concentrations of MS, with 0.1% DMSO as a control. Images were collected under a stereomicroscope at 54 hpf to record embryonic development. A Tg(fli1a:EGFP) transgenic line was used for pharmacological experiments and mechanism validation in the MS experiment. Two researchers participated in the embryo experiments: one injected embryos and administered MS to zebrafish embryos, while the other recorded the embryonic phenotype, performed statistical analysis, and took photographs without clearly defining experimental groups.

[0036] Mouse experiment: 12 NSG mice were included in the experiment. TM Mice. These mice were divided into three groups: DDX24 NC, DDX24 deficient, and DDX24 deficient + MS. A premixed matrix gel containing pretreated endothelial cells, PBS (0.2 ml), and vascular endothelial growth factor (VEGF; 500 ng / ml) was mixed. The mixed matrix gel was then subcutaneously injected into 6-8 week old NSG mice. TM The lateral ventral portion of mice was injected (0.2 ml per mouse). Fourteen days post-injection, the matrix plugs were harvested and photographed. Endothelial cells were stained with the marker CD31 by immunofluorescence of 10 μm sections embedded in OCT. Three researchers participated in the embryo experiments: one researcher administered MS via injection, and two researchers performed dissection, photography, and immunofluorescence observation.

[0037] Gene knockdown and plasmid overexpression: DDX24 or S100A8 siRNA was transfected with LTX (Invitrogen) to generate DDX24 or S100A8 knockdown cells. For plasmid transfection, Lipo3000 was used, transfecting 10 μg of plasmid per 10 cm culture dish, transfecting 70% confluence cells. Human DDX24 fused with the C-terminal Flag tag and S100A8 fused with the Myc tag were obtained from IGE BIO. Cell lysates were collected 12 h after MS treatment and analyzed by Western blot.

[0038] Immunoprecipitation and protein blotting: Follow the instructions in the manual, using Pierce... TMThe Co-IP Kit uses anti-DDX24 antibody to detect endogenous DDX24 Co-IP. Normal rabbit IgG is used as a control. Exogenous Co-IP complexes are detected using anti-Flag or anti-Myc antibody. Samples are then boiled and analyzed by Western blotting. For Western blotting, samples are separated by SDS-PAGE and then electroblotted onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane is blocked in TBST (0.1% Tween 20, 50 mM Tris-HCl (pH 7.4), and 150 mM NaCl) containing 5% skim milk for 1 hour, and then incubated overnight at 4°C with a primary antibody. The next day, the membrane is incubated with the corresponding secondary antibody and detected using ECL reagent.

[0039] Immunofluorescence microscopy: Cells were seeded into 15 mm glass-bottom confocal culture dishes and fixed in 4% paraformaldehyde for 15 minutes at room temperature, followed by staining. The slides or culture dishes were then incubated in TBST (TBS containing 0.1% Triton X-100) for 10 minutes, blocked at room temperature (10% goat serum + 1% BSA + 0.05% Tween 20) for 1 hour, and then incubated overnight at 4°C with various primary antibodies. Alexafluor 488-conjugated goat anti-rabbit (BOSTER, BA1126) and Alexafluor 555-conjugated goat anti-mouse IgG (Bioss, bs-0296G-AF555) were used. Slides were mounted with DAPI-conjugated anti-quenching mounting medium (Solarbio, S2110) before analysis using a Zeiss 880 confocal laser scanning microscope.

[0040] Purification of recombinant proteins: The full-length human S100A8 (S100A8-wt) transcript variant gene (NCBI reference sequence: NP_0029559.2) or the S100A8 mutant was fused with a 6x His tag and subcloned into the pET-28a(+) vector. The plasmid was transformed into BL21(DE3) competent cells (11804ES80, Yeasen) by induction at 16°C for 16 h with 200 μM IPTG. Cells were harvested by centrifugation. All purification steps were performed on ice. The cell pellet was resuspended and sonicated in lysis buffer consisting of buffer A (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 25 mM imidazole) and loaded onto a Ni-NTA column washed with buffer B (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 50 mM imidazole-HCl, pH 8.0). The bound protein was eluted from the column using buffer C (20 mM Tris-HCl, pH 8.0, 500 mM NaCl, 500 mM imidazole-HCl, pH 8.0). The desired protein was then purified by size exclusion chromatography in HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid) buffer (10 mM HEPES, pH 8.0; 150 mM NaCl). For the DDX24 protein, the experiments were performed according to existing literature.

[0041] Cellular thermal shift assay (CETSA): HUVEC or HEK293T cells were cultured in 10 cm culture dishes, treated with DMSO or MS (10 μM) for 1 hour, and resuspended in PBS supplemented with protease inhibitors. Samples were aliquoted into eight portions and heat-treated at 41, 44, 47, 50, 53, 56, 59, and 63 °C for 5 minutes each, followed by three freeze-thaw cycles in liquid nitrogen. The samples were then centrifuged to obtain the supernatant. An SDS-PAGE loading buffer was added. Samples were analyzed using Western blotting.

[0042] Drug affinity-responsive target stability (DARTS) assay: HUVEC or HEK293T cells were lysed in RIPA buffer at a ratio of 4–6 mg / mL. The lysis buffer was then treated with different concentrations of MS or DMSO at room temperature for 1 h. Cells were rapidly proteolysed for 10 min at 25 °C with 1 μg / mL Pronase E (Roche, 10165921001), followed by incubation with SDS-PAGE loading buffer at 95 °C for 10 min. Proteins and proteolytic fragments were separated by SDS-PAGE and detected by Western blotting using anti-S100A8 antibody (R&D, MAB4570) or anti-Myc antibody (Vazyme, RA1005-01).

[0043] Computer modeling: Molecular docking studies were performed using Schrodinger 2018.1 / Glide. The crystal structure of S100A8 (PDB code: 5HLO) was downloaded from the RCSB protein database and prepared using the protein preparation wizard panel implemented in Maestro 10.5: water molecules were removed, and missing hydrogens and residues were added. Binding sites were then defined; compounds were generated in MS format using Maestro 13.1 and then docked to the S100A8 binding sites using Glide in XP mode. All docking results were processed using Maestro 13.1 and PyMOL software.

[0044] Surface plasmon resonance (SPR) assay: The interaction between immobilized MS and His-S100A8 or DDX24 was detected using a Biacore T200 instrument. Experiments were performed as described above.23 Briefly, purified DDX24 or S100A8 protein was dissolved in 0.05 mg / ml of 10 mM NaOAc (sodium acetate) buffer (pH 4.5) and then immobilized on a CM5 chip of approximately 10,000 response units (RUs) via amine coupling according to the manufacturer's instructions (GE Healthcare). The protein sample was first flowed at 30 μL / min for 300 seconds to associate, then flowed with flow buffer for 300 seconds to dissociate, and then regenerated at 30 μL / min for 60 seconds to obtain the binding curve. The MS-containing masterbatch was diluted with DMSO-free PBS using the isocratic dilution method to obtain MS protein (S100A8 or DDX24) RUs. The dissociation constants (Kd) of compounds and proteins are fitted using a standard 1:1 combination model, and then obtained by global fitting of the kinetic data of compound gradient concentrations.

[0045] mRNA stability assay: Cells were pretreated with MS (TargetMol, A4448-5 mg) for 12 hours with or without the addition of actinomycin-D to a final concentration of 50 μg / mL. Total mRNA was extracted and quantified by real-time RT-PCR at 3, 6, and 9 h post-treatment. Results were normalized relative to 0 h.

[0046] Cell confluence assay: After cells reached 60%–70% confluence, transfection was prepared. First, cells were knocked down using DDX24 siRNA, and the medium was replaced with fresh medium after 6 hours. 24 hours later, cells were infected with viral supernatant containing S100A8-Myc, and the medium was replaced with fresh medium after 6 hours. 50 μL of matrix gel was spread onto pre-chilled 96-well plates and incubated at 37°C for 30 minutes. Cells were digested and collected for counting. 100 μL of HUVEC cell suspension (approximately 1.3 million cells) was added to each well. DMSO medium and MS medium (2.5 μmol / L) were added separately, and the plates were incubated at 37°C for approximately 6–8 hours to observe angiogenesis. Photographs were taken and recorded. The experiment was repeated three times.

[0047] Cell migration assay: After cells reached 60%–70% confluence, transfection was prepared. First, cells were knocked down using DDX24 siRNA, and the medium was replaced with fresh medium after 6 hours. 24 hours later, cells were infected with viral supernatant containing S100A8-Myc, and the medium was replaced with fresh medium after 6 hours. 48 hours after infection, cells were digested and resuspended in serum-free medium, adjusting the cell density to (7–8) × 10⁻⁶ cells / year. 5 Under aseptic conditions, remove the Millicell insertion chamber and place it into a sterile 24-well plate. Add 600 μL of DMSO or MS (2.5 μmol / L) of the same concentration to each well. Place 300 μL of cell suspension into the upper chamber of a Transwell plate. Incubate the plate with the cell suspension under normal conditions (5% CO2, 95% O2) for 12 h. Then remove the insertion chamber, gently wipe away the cells on the inner surface of the upper chamber filter membrane with a cotton swab, place the entire insertion chamber into a 24-well plate, fix with 95% ethanol for 15 min, and stain with 0.1% crystal violet for 20 min. Count the number of cells that have passed through the membrane. Repeat the entire experiment three times.

[0048] Example 1: Montelukastner can eliminate DDX4 deficiency-induced vascular malformations.

[0049] First, the role of MS in DDX24-deficient cells was investigated in vitro. Cell migration results showed that MS reversed the increased cell migration after DDX24 knockdown. Figure 1 A and 1B). Similarly, tube formation assays reflect the results of cell migration ( Figure 1 (C and 1D). However, MS treatment did not affect the proliferative capacity of HUVECs. These data suggest that MS inhibits DDX24-deficiency-induced angiogenesis.

[0050] To investigate the effects of MS on in vivo intersegmental vessels (MOVLD), transgenic zebrafish Tg(fli1a:EGFP) embryos were injected with ddx24 MO in 1–4 cells. After treatment with DMSO or MS at different concentrations of 2.5, 5, and 10 μM for 48 h, we found that the resulting excessively branched intersegmental vessels (ISVs) phenotype was similar to that of MOVLD patients. Figure 1 E-1G). As expected, the abnormalities caused by Ddx24 deficiency were salvaged by MS treatment. Based on these results, MS may upregulate DDX24 protein expression. In fact, after MS treatment, the DDX24 protein level increased in both DDX24-deficient HUVECs and zebrafish embryos. Figure 1 H and 1I).

[0051] To further validate the effect of MS on VM in mammalian models, we performed Matrigel thrombus assays in a mouse model and demonstrated that excessive angiogenesis caused by DDX24 deficiency could be completely eliminated by MS treatment. Figure 1 J and 1K). In summary, the above results indicate that DDX24 deficiency leads to excessive angiogenesis and ISV superbranching in zebrafish endothelial cells, which can be alleviated by MS treatment.

[0052] Example 2S100A8 is a direct target of Monteluka.

[0053] The classic mechanism of MS is targeting a known protein—cysteyl leukotriene receptor 1 (CysLTR1). To test whether the effects of MS on endothelial cells depend on CysLTR1, the receptor was removed using siRNA and analyzed by Western blotting. Figure 2 A) Verification. Knockdown of CysLTR1 inhibits cell migration and tube formation ( Figure 2 (B and 2C). Unexpectedly, MS treatment further reduced cell migration, suggesting that MS may exert its anti-angiogenic function by acting as a substitute target for CysLTR1 or by acting beyond CysLTR1.

[0054] To investigate protein targets that directly bind to MS, we conjugated montelukast with biotin and performed pull-down experiments in HUVEC, followed by mass spectrometry analysis. Figure 3 A). We identified 28 biotin-MS-targeting proteins in two biological replicates, which were primarily enriched in the regulation of tissue migration and binding pathways. On the other hand, we also examined proteins that can bind to DDX24 using a pull-down assay, as DDX24 is likely a downstream component of the MS target. Figure 3 B). By overlaying the proteins detected by the two methods above, only one protein, S100A8, located in the cytoplasm or membrane, is obtained. Figure 3(C) This protein is known for its function in regulating macrophage migration and is closely related to inflammatory processes and immune responses. Therefore, we will focus our further research on S100A8.

[0055] To verify that S100A8 is the true binding partner of MS, cell thermal shift assays were performed. Compared with S100A8 treated with DMSO, S100A8 treated with MS showed greater stability at higher temperatures. Figure 3 D). Furthermore, assessment using the Drug Affinity-Responsive Target Stability (DARTS) assay revealed increased resistance of S100A8 to Pronase E-induced proteolysis after MS treatment. Figure 3 E). To further demonstrate the binding affinity between MS and S100A8, surface plasmon resonance (SPR) detection was applied after purifying S100A8 and DDX24 proteins. Figure 4 A). Data shows that the dissociation constant (Kd) of S100A8 is 1.07 μM ( Figure 3 F), while the Kd of DDX24 is 15.79 μM ( Figure 4 B).

[0056] Furthermore, to determine the MS binding region on the S100A8 protein (PDB code: 5HLO-B), we performed molecular docking using Schrödinger software. The docking model showed that the carboxyl group of compound MS, acting as a hydrogen bond donor, can form a hydrogen bond with SER11 located in Helix1 at a distance of [missing information]. ( Figure 3 G). In addition to strong hydrogen bonding interactions, the quinoline ring and benzene ring can also interact hydrophobically with residues PHE67, PHE71, and VAL75 at the S100A8 binding site. When the Helix1 domain in S100A8 is deleted (S100A8-MUT), it cannot bind to MS. Figure 3 H and 3I, Figure 4 C). In summary, our multidimensional data suggest that S100A8 is a direct protein target for MS.

[0057] Example 3: MS combined with S100A8 inhibits angiogenesis

[0058] To confirm whether MS inhibits angiogenesis in a way that depends on S100A8, we knocked down S100A8 in HUVECs and validated this by Western blotting and RT-qPCR. Figure 5 A and 5B). We found that MS treatment did not alter cell migration and tube formation in the absence of S100A8 (A and B). Figure 6(A~6D). To further confirm that the effect of MS on DDX24 expression depends on S100A8, we demonstrated that S100A8 deficiency eliminated the accumulation of DDX24 protein in the cytoplasm after MS treatment, using Western blotting and IF analysis ( Figure 6 E and 6F). Knockdown of S100A8 alleviated MS-induced decrease in DDX24 protein ubiquitination ( Figure 6 G). In terms of cell function, transfection with S100A8-WT can rescue the role of MS in S100A8-deficient HUVECs (G). Figure 6 H~6I). In contrast, transfection with S100A8-MUT failed to restore the effect of MS (H~6I). Figure 6 (J~6K). Therefore, these data indicate that S100A8 is crucial for the effects of MS on DDX24 regulation and angiogenesis.

[0059] Example 4: MS stabilizes DDX24 by promoting the Q-motif interaction between S100A8 and DDX24.

[0060] Next, we attempt to further investigate the MS-mediated interaction between S100A8 and DDX24. S100A8 can pull down DDX24 (… Figure 7 A). Furthermore, co-IP and IF experiments showed that higher DDX24 levels were associated with higher binding of S100A8 protein in the cytoplasm, and MS enhanced this interaction. Figure 7 B and 7C; Figure 8 A and 8B). To further determine the DDX24 region interacting with S100A8, different DDX24 domains were truncated and their binding with S100A8 was tested. Figure 7 D). Co-IP data show that DDX24 mainly interacts with S100A8 through the Q motif (D). Figure 7 E). Therefore, we transfected cells with full-length (FL), N2 (with Q motif), or C2 (without Q motif) DDX24 protein and examined the colocalization of these fragments with S100A8. IF results confirmed that the Pearson correlation coefficient between DDX24 N2 and S100A8 was the same as that between DDX24 FL and S100A8. Figure 7 F and 8C), while co-location between DDX24C2 and S100A8 is severely hampered ( Figure 7 G). Overall, these data suggest that MS primarily promotes the interaction between DDX24 and S100A8 through the Q motif of DDX24.

[0061] Example 5: S100A8 enhances the therapeutic effect of MS on DDX4 deficiency-induced vascular malformations.

[0062] We have demonstrated that S100A8 is a target for treating DDX4 deficiency-induced vascular malformations in MS. Without S100A8, MS has no therapeutic effect on DDX4 deficiency-induced vascular malformations. Next, we will examine the therapeutic effect of expressing S100A8 on MS.

[0063] The results showed that, Figure 9 Figure A shows that MS can inhibit the increase in endothelial migration induced by DDX24 knockdown (comparison of the first three columns), but overexpression of S100A8 can reverse the increase in endothelial migration caused by DDX24 knockdown (comparison of the second and fourth columns in the presence of MS); based on S100A8 overexpression, MS can further inhibit the increase in endothelial migration induced by DDX24. The results indicate that S100A8 can enhance the therapeutic effect of MS.

[0064] like Figure 9 B shows that MS can inhibit the increase in endothelial tube formation induced by DDX24 deficiency (comparison of the first three columns); overexpression of S100 can reverse the increase in endothelial tube formation caused by DDX24 knockdown (comparison of the second and fourth columns); overexpression of S100A8 can inhibit the increase in endothelial tube formation caused by DDX24 deficiency (in the presence of MS), that is, S100A8 can enhance the efficacy of MS treatment (comparison of the third and fifth columns).

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of S100A8 in the preparation of functional products for treating vascular malformations caused by DDX24 knockdown with sodium montelukast.

2. The application according to claim 1, characterized in that, The functional product serves as an adjuvant for montelukast sodium in the treatment of vascular malformations caused by DDX24 knockdown.

3. The use of S100A8 and montelukast sodium as a pharmaceutical composition in the preparation of a drug for treating vascular malformations caused by DDX24 knockdown.

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