Screening method for using endophenic acid as interleukin-8 antagonist and drug application of endophenic acid as interleukin-8 antagonist

Abstract

The invention relates to a method for screening an interleukin-8 antagonist and a drug application of the interleukin-8 antagonist, and the method for screening the interleukin-8 antagonist comprises the following steps: S1, customizing a specific interleukin-8 as a target, and customizing an FDA drug library as a drug molecule library for later use; s2, performing high-throughput molecular screening in a drug molecule library by using the target, and screening specific drug molecules capable of being combined with the target from the drug molecule library; and S3, detecting the antagonism effect of the specific drug molecule on the target, and performing target antagonism agent-effect evaluation on the specific drug molecule.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, and in particular to a screening method for embezzic acid as an interleukin-8 antagonist and its pharmaceutical application. Background Technology

[0002] Interleukin-8 (IL-8), also known as chemokine CXCL8, is a small protein composed of 72 amino acid residues with a molecular weight of approximately 8 kDa. Its structure contains typical CXC chemokine characteristics, namely, two cysteine ​​residues separated by an amino acid. IL-8 is an important inflammatory cytokine, primarily produced by non-immune cells such as endothelial cells, epithelial cells, and fibroblasts, as well as immune cells such as monocytes, macrophages, and neutrophils, under inflammatory stimulation. IL-8 expression is strictly dependent on inflammatory signaling regulation; its expression is extremely low under normal physiological conditions, and rapidly upregulated after inflammatory stimulation.

[0003] IL-8, a key chemokine, specifically binds to CXCR1 / 2 receptors on the surface of target cells, activating downstream signaling pathways and recruiting and activating neutrophils to participate in host defense and tissue repair processes, playing a crucial role in inflammatory responses and diseases. When the body is infected or injured, damaged cells rapidly secrete IL-8, causing neutrophils to migrate rapidly to inflamed tissues and activating various effector functions of neutrophils, enhancing the body's ability to clear pathogens and kill bacteria. During inflammation resolution and tissue repair, IL-8 can promote the proliferation and migration of vascular endothelial cells, participating in angiogenesis, regulating fibroblast activity, promoting collagen synthesis, and participating in tissue repair processes.

[0004] In autoimmune inflammatory diseases, IL-8 participates in tissue damage by excessively recruiting neutrophils. In rheumatoid arthritis patients, IL-8 levels are significantly elevated in the synovial fluid and tissue, and are positively correlated with the degree of neutrophil infiltration. IL-8-mediated neutrophil infiltration can release large amounts of proteases and reactive oxygen species, leading to the destruction of articular cartilage and bone tissue, exacerbating joint inflammation and functional impairment. Among these, proteases include matrix metalloproteinases. In psoriatic lesions, IL-8 expression is significantly upregulated, participating in the formation of psoriatic plaques by recruiting neutrophils to the epidermis, promoting abnormal keratinocyte proliferation, and maintaining the chronic inflammatory state of the lesions.

[0005] Given the crucial role of IL-8 in disease, biologics primarily composed of IL-8 inhibitors are an important component of IL-8 antagonists. Antibody drugs alleviate disease symptoms by blocking the interaction between IL-8 and CXCR1 / 2, inhibiting the accumulation of immune cells and inflammatory factors. Antibody therapy can replace hormone therapy; when combined with glucocorticoids, it can reduce the dosage of hormones and decrease the incidence of adverse drug reactions. Inflammatory cytokines, cell surface molecules, and their mediated signaling pathways participate in the pathological process of autoimmune diseases; targeted biologics targeting cytokines, receptors, and signaling molecules have seen rapid development in recent years.

[0006] IL-8 plays a crucial role in various diseases, but antibody therapy targeting IL-8 has certain limitations. Biologic antibody drugs carry certain side effects or treatment risks. Acute reactions following monoclonal antibody injection can be caused by multiple mechanisms, including acute anaphylaxis (IL-8-mediated) and anaphylactic-like reactions, serum sickness, tumor lysis syndrome, and cytokine release syndrome. Clinical manifestations include local skin reactions at the injection site, fever, and flu-like syndrome. Humanization of antibody drugs does not completely eliminate immunogenicity; even the CDR region of humanized antibodies retains strong immunogenicity, generating anti-idiotype antibodies and leading to drug inactivation. Furthermore, antibody drugs are unstable and their structure is prone to change. The pharmacokinetic properties of antibody drugs are also unfavorable.

[0007] Currently, the treatment of various IL-8-mediated diseases relies on biological antibody drugs or hormone drugs with significant side effects. These IL-8-mediated diseases seriously affect people's health and lives. Therefore, it is of great importance and social significance to develop drugs that can antagonize IL-8 molecules with good efficacy and fewer side effects. Summary of the Invention

[0008] Therefore, it is necessary to provide screening methods for embezzic acid as an interleukin-8 antagonist and its pharmaceutical applications.

[0009] One aspect of this invention provides the use of embenic acid in the preparation of interleukin-8 antagonists.

[0010] On the other hand, a method for screening interleukin-8 antagonists is provided, which includes the following steps:

[0011] S1. Customize a specific interleukin-8 as a target and customize an FDA drug library as a drug molecule library for backup.

[0012] S2. High-throughput molecular screening of drug molecules using targets to screen specific drug molecules that can bind to targets from the drug molecule library.

[0013] S3. Detect the antagonistic effect of the specific drug molecule on the target, and evaluate the efficacy of the specific drug molecule in antagonizing the target.

[0014] Preferably, in step S1, 3067 FDA-approved compound drugs are prepared and dissolved in DMSO at 1 mg / ml; an IL-8 protein target is prepared, the purity of which is determined to be 95% by SDS-PAGE; the IL-8 protein target is diluted with deionized water in a gradient concentration; wherein, the amino acid sequence of the IL-8 protein target is shown in SEQ ID NO: 1.

[0015] Preferably, in step S2, the FDA drug library is assembled into a microfluidic chip and installed in a PlexArray HT SPRi microarray analyzer; then, the IL-8 target to be tested is injected sequentially according to the concentration gradient; finally, the collected signal data is analyzed using the data analysis software Data Processor Stand Alone, and the results are sorted by molecular affinity to select the molecular compounds that meet the requirements from the drug molecules that can bind to the target.

[0016] Preferably, the drug molecule screened by the screening method is embenic acid.

[0017] Preferably, before sequentially injecting the IL-8 target to be tested according to the concentration gradient, a positive sample is used to pre-test the microfluidic chip, and the chip quality is verified based on whether a positive response signal of the positive sample is detected.

[0018] Preferably, the positive sample is rapamycin.

[0019] Preferably, in step S3, the antagonistic effect of embezzic acid on IL-8 is detected using a human interleukin-8 (IL-8) ELISA kit. The antagonistic effect is evaluated by detecting the absorbance using an ELISA reader, establishing a standard curve, calculating the detection concentration, and calculating the antagonistic rate based on the detection concentration.

[0020] Preferably, the antagonism rate is the quotient of the detected concentration and the initial concentration.

[0021] On the other hand, an interleukin-8 antagonist is also provided, wherein the antagonist is embezzic acid.

[0022] This invention provides a method for high-throughput screening of drug molecules that can bind to IL-8 in the FDA drug library using IL-8 as a target, and for verifying their antagonistic efficiency at the in vitro molecular level. This method screens out a small molecule drug, embezzic acid, that can antagonize IL-8, providing a new approach for studying the function of IL-8. Attached Figure Description

[0023] The above and other objects, features, and advantages of the invention will become clearer through a more detailed description of the preferred embodiments illustrated in the accompanying drawings. The same reference numerals denote the same parts throughout the drawings, and the drawings are not intentionally drawn to scale with actual dimensions; the focus is on illustrating the gist of the invention.

[0024] Figure 1 The graph shows the embenic acid signal curves collected when IL-8 protein gradient concentrations were 2000 nM, 1000 nM, 500.0 nM, 250.0 nM, 125.0 nM, 62.5 nM, 31.2 nM and 15.6 nM.

[0025] Figure 2 The chromatograms show the dose-effect analysis of embezzic acid at concentrations of 0 pg / L, 16.65 pg / L, 83.26 pg / L, 416.32 pg / L, 2081.61 pg / L, 10408.07 pg / L and 52040.33 pg / L.

[0026] Figure 3 This is the molecular structure diagram of embenic acid. Detailed Implementation

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0028] like Figures 1-3 As shown, a method for screening interleukin-8 antagonists and the antagonist themselves include the following steps:

[0029] Step 1: A drug molecule library consisting of 3067 FDA-approved chemical drugs was established and stored after dissolution in DMSO at a concentration of 1 mg / ml. Interleukin-8 was customized as the IL-8 protein target, with a purity of 95% as determined by SDS-PAGE. IL-8 was serially diluted with deionized water to 2.0 μM, 1000 nM, 500.0 nM, 250.0 nM, 125.0 nM, 62.5 nM, 31.2 nM, and 15.6 nM. The amino acid sequence of the IL-8 target is shown in SEQ ID NO: 1.

[0030] The amino acid sequence of IL-8 is as follows:

[0031] SEQ ID NO: 1:

[0032] 1AVLPRSAKELRCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPK

[0033] 61ENWVQRVVEKFLKRAENS

[0034] Step 2: Perform high-throughput molecular screening in the drug molecule library using the target to screen for specific drug molecules that can bind to the target.

[0035] High-throughput molecular screening targeting IL-8: FDA drug library chips were assembled into microfluidic chips and installed in a PlexArray HT SPRi microarray analyzer. The chip quality was verified using rapamycin as a positive sample. The target proteins were injected sequentially according to the concentration gradient described above. Finally, the collected signal data were analyzed using Data Processor StandAlone software, and the results were ranked according to molecular affinity.

[0036] Step 3: Detect the antagonistic effect of this specific drug molecule on the target, and evaluate the efficacy of this specific drug molecule in antagonizing IL-8. The selected drug molecule is embezzic acid.

[0037] The screened drug molecules were validated at the drug molecule level: the antagonistic effect of embezzic acid on IL-8 was evaluated using the Human Interleukin-8 (IL-8) ELISA kit (Shenzhen Zike). The absorbance was measured using an ELISA reader, and the antagonistic rate was calculated based on the detected concentration: Antagonistic rate = 100% - (detected concentration / initial concentration).

[0038] like Figure 1 and 2 As shown, embenic acid has a novel application as a human interleukin-8 antagonist.

[0039] In a preferred embodiment, high-throughput screening of the FDA drug library chip is performed using IL-8 as the target, and the steps are as follows:

[0040] (1) Take out the prepared FDA drug library chip, place it in 1% BSA solution for 2 h to seal it, wash the chip with pure water and dry it before assembling it into a microfluidic chip.

[0041] (2) 1 mg / ml of interleukin-8 (IL-8) protein was serially diluted with PBST buffer.

[0042] (3) The microfluidic chip was installed in the PlexArray HT SPRi microarray analyzer. First, a positive sample of FKBP12 with a concentration of 100 nM was injected at a flow rate of 1 μl / sec for 300 sec. The positive response signal of rapamycin was detected to verify the chip quality.

[0043] (4) Wash with PBST buffer at a flow rate of 1 μl / s for 300 sec. Inject the IL-8 protein to be tested sequentially according to the gradient concentration at a flow rate of 1 μl / sec for 300 sec. Dissociate with PBST buffer at a flow rate of 1 μl / s for 300 sec. Repeat the above steps until all concentrations of IL-8 protein have been injected. Collect signal data.

[0044] (5) The chip was regenerated and stored after binding for 120 s and dissociating for 180 s with Gly-HCl buffer (pH=2.0) at a flow rate of 1 μl / s.

[0045] (6) The collected signal data were analyzed using the data analysis software Data Processor Stand Alone, and the results were sorted by molecular affinity. The small molecule compound embenic acid was screened.

[0046] As attached Figure 1 As shown, with increasing IL-8 protein concentration, the data collected by the DataProcessor Stand Alone analysis software became stronger, and embenic acid showed the highest affinity among all small molecules, making it a candidate molecule for further research. Figure 3 The molecular structure of embenic acid.

[0047] In a preferred embodiment, the efficacy analysis of endocyanic acid antagonizing IL-8 was performed, and the steps are as follows:

[0048] (1) Add 50 μl of 10 μg / ml IL-8 protein to an EP tube, add different concentrations of embezzic acid, and incubate at 37℃ for 1 h.

[0049] (2) Add 100 μl of protein small molecule mixture or standard and 100 μl of HRP-labeled IL-8 antibody to the pre-coated microwells, incubate at 37°C for 1 h, discard the solution, wash 5 times, and develop TMB color.

[0050] (3) Measure the absorbance at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, establish a standard curve, and then calculate the detection concentration.

[0051] The materials and reagents used were as follows: FDA drug library chip purchased from Selleck Chemicals; interleukin-8 purchased from MCE; PBST buffer purchased from Thermo Fisher Scientific; Gly-HCl buffer purchased from Thermo Fisher Scientific; Human interleukin-8 (IL-8) ELISA kit purchased from Shenzhen Zike Biotechnology Co., Ltd.; and embezzled acid purchased from MedChemexpress Biotechnology Co., Ltd. (USA).

[0052] The antagonism rate was calculated based on the detected concentration: Antagonism rate = 100% - (detected concentration / initial concentration). The results are shown in Table 1 below.

[0053] Table 1. Antagonism rates of interleukin-8 at different concentrations of embezzled acid

[0054] IL-8: Targeted drugs (quantity ratio) 1:0 1:01 1:05 1:25 1:125 1:625 1:3125 Enbecic acid concentration (pg / L) 0 16.65 83.26 416.32 2081.61 10408.07 52040.33 IL-8 detection concentration (pg / ml) 55.80 54.90 52.64 54.19 50.39 46.66 14.63 Antagonism rate 0.00% 1.60% 5.65% 2.88% 9.69% 16.37% 73.77%

[0055] refer to Figure 2 Analysis of the results showed that the antagonistic effect of embezzic acid on IL-8 protein was 73.77% when the drug concentration was 52.04 μg / L.

[0056] In summary, this invention targets the IL-8 protein, performs high-throughput screening of drug molecules in the FDA drug library that can bind to IL-8, and verifies their antagonistic efficiency at the in vitro molecular level. A molecule that can antagonize IL-8 was screened out, and the antagonistic efficiency of the screened embezzic acid is significantly higher than that of ordinary antagonists.

[0057] Embelin is a quinone compound isolated from Ardisia crenata. It is a benzoquinone compound that can penetrate cells and possesses anticancer and anti-inflammatory activities. Ardisia crenata is a traditional Chinese medicine used to relieve coughs and asthma, processed from the dried whole plant of the Ardisia crenata plant. It can resolve phlegm, relieve asthma, clear damp heat from the body, and improve blood circulation, showing therapeutic effects on common ailments such as colds, coughs, and joint pain. Currently, no reports have been found on the content and pharmacokinetic data of embelin in Ardisia crenata.

[0058] In the field of anti-inflammatory and immunomodulatory effects, embezzic acid can reduce inflammatory responses by inhibiting the NF-κB signaling pathway, blocking its nuclear translocation and the expression of downstream inflammatory factors such as IL-6 and TNF-α. At the same time, it can regulate macrophage polarization, inhibit the pro-inflammatory phenotype M1, and promote the anti-inflammatory phenotype M2, thus showing potential therapeutic value in chronic inflammatory disease models such as rheumatoid arthritis and enteritis.

[0059] In terms of anti-tumor activity, embezzic acid can target X-linked inhibitor of apoptosis protein (XIAP) to relieve its inhibitory effect on the Caspase family and induce tumor cell apoptosis. At the same time, it can inhibit proliferation-related signaling pathways such as PI3K / Akt and MAPK, reduce tumor cell proliferation activity, and inhibit vascular endothelial growth factor (VEGF)-mediated tumor angiogenesis. It has shown anti-proliferation, pro-apoptosis and metastasis-inhibiting effects in various tumor models such as lung cancer, liver cancer and breast cancer.

[0060] In addition to its antioxidant, mitochondrial uncoupling, and antifertility properties, enoxaparin also exhibits antidiabetic, anticonvulsant, anti-anxiety, and antimicrobial activities. Ibexaparin is an effective neuroprotective agent and can also be used as an adjunct therapy for stroke.

[0061] This invention targets IL-8, performs high-throughput screening of drug molecules in the FDA drug library that can bind to IL-8, and verifies their antagonistic efficiency at the in vitro molecular level. It screens out a small molecule drug, embenic acid, that can antagonize IL-8, and the screened small molecule drug embenic acid provides a new approach for studying the function of IL-8.

[0062] The above are merely preferred embodiments of the present invention and do not limit the scope of the patent. Any equivalent structural or procedural transformations made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. Use of embenic acid in the preparation of interleukin-8 antagonists.

2. A method for screening interleukin-8 antagonists, characterized in that, The screening method for interleukin-8 antagonists includes the following steps: S1. Customize a specific interleukin-8 as a target and customize an FDA drug library as a drug molecule library for backup. S2. High-throughput molecular screening of drug molecules using targets to screen specific drug molecules that can bind to targets from the drug molecule library. S3. Detect the antagonistic effect of the specific drug molecule on the target, and evaluate the efficacy of the specific drug molecule in antagonizing the target.

3. The screening method as described in claim 1, characterized in that, In step S1, 3067 FDA-approved compound drugs were customized and dissolved and stored in DMSO at 1 mg / ml; an IL-8 protein target was customized, and its purity was determined to be 95% by SDS-PAGE; the IL-8 protein target was diluted with deionized water in a gradient concentration; wherein, the amino acid sequence of the IL-8 protein target is shown in SEQ ID NO:

1.

4. The screening method as described in claim 1, characterized in that, In step S2, the FDA drug library is assembled into a microfluidic chip and installed in the PlexArray HT SPRi microarray analyzer; then, the IL-8 target to be tested is injected sequentially according to the concentration gradient; finally, the collected signal data is analyzed using the data analysis software Data Processor Stand Alone, and the results are sorted by molecular affinity to select the molecular compounds that meet the requirements from the drug molecules that can bind to the target.

5. The screening method as described in claim 1, characterized in that, The drug molecule screened by the aforementioned screening method is embenic acid.

6. The screening method as described in claim 1, characterized in that, Before sequentially injecting the IL-8 target to be tested according to the concentration gradient, the microfluidic chip is pre-tested with positive samples. The chip quality is verified by whether a positive response signal is detected from the positive sample.

7. The screening method as described in claim 6, characterized in that, The positive sample was rapamycin.

8. The screening method as described in claim 1, characterized in that, In step S3, the antagonistic effect of embezzled acid on IL-8 is detected using a human interleukin-8 (IL-8) ELISA kit. The antagonistic effect is evaluated by detecting the absorbance using an ELISA reader, establishing a standard curve, calculating the detection concentration, and calculating the antagonistic rate based on the detection concentration.

9. The screening method as described in claim 8, characterized in that, The antagonism rate is the quotient of the detected concentration and the initial concentration.

10. An interleukin-8 antagonist, characterized in that, The antagonist is embezzic acid.

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