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CN-121978063-A - Screening method and application of pulegone serving as interleukin-8 antagonist

CN121978063ACN 121978063 ACN121978063 ACN 121978063ACN-121978063-A

Abstract

The invention relates to a screening method and application of an interleukin-8 antagonist, wherein the screening method of the pulegone interleukin-8 antagonist comprises the following steps of S1, customizing a drug molecule library, customizing interleukin-8 as a target, S2, screening high-flux molecules in the drug molecule library by using the target, screening drug molecules capable of being combined with the interleukin-8 target from the drug molecule library, S3, verifying in vitro antagonistic activity of the drug molecules screened in the step S2, thereby detecting antagonistic effect of the specific drug molecules on the target effect, and evaluating the antagonistic effect of the specific drug molecules on interleukin-8.

Inventors

  • WANG HAI
  • TANG LEI

Assignees

  • 广州创瑞健康科技有限公司

Dates

Publication Date
20260505
Application Date
20251231

Claims (10)

  1. 1. A method of screening for an interleukin-8 antagonist comprising the steps of: s1, customizing a drug molecule library, and customizing interleukin-8 as a target for later use; S2, performing high-flux molecular screening in a drug molecular library by using a target, and screening drug molecules capable of combining with interleukin-8 targets from the drug molecular library; s3, verifying the in-vitro antagonistic activity of the medicine molecules screened in the step S2, thereby detecting the antagonistic effect of the specific medicine molecules on the target effect, and evaluating the antagonistic effect of the specific medicine molecules on interleukin-8.
  2. 2. The screening method of claim 1, wherein the drug molecular library is an FDA approved 3067 compound drug library.
  3. 3. The screening method according to claim 1 or 2, wherein in step S1, the compound drug in the drug molecule library is dissolved and stored in DMSO at a concentration of 1 mg/mL, the purity of the interleukin-8 target is 95% or more, and the interleukin-8 target is subjected to gradient concentration dilution by deionized water, wherein the amino acid sequence of the interleukin-8 target is shown as SEQ ID NO. 1.
  4. 4. The screening method according to claim 1, wherein in step S2, high throughput screening is performed using a surface plasmon resonance imaging technique, specifically comprising: S21, fixing and assembling the drug molecule library into a microfluidic chip; S22, mounting the microfluidic chip in a PlexArray HT SPRi microarray analyzer; s23, sequentially sampling the interleukin-8 target to be tested according to a concentration gradient; s24, analyzing the collected signal data of the detection binding signals through data analysis software Data Processor Stand Alone, sorting the molecular affinity of the analysis results, and screening out the compound drug bound with the target.
  5. 5. The screening method according to claim 4, wherein the chip quality is verified by detecting a positive response signal of the positive sample in advance using a positive control sample injection before proceeding to step S23.
  6. 6. The screening method of claim 5, wherein the positive control sample is rapamycin.
  7. 7. The screening method according to claim 1, wherein the antagonism of interleukin-8 by the compound is evaluated by enzyme-linked immunosorbent assay in step S3.
  8. 8. The screening method according to claim 7, wherein the antagonistic ratio is calculated by the formula of antagonistic ratio= (1-detection concentration/initial concentration) ×100%.
  9. 9. An interleukin-8 antagonist characterized by, the antagonist is pulegone.
  10. 10. Use of pulegone in the manufacture of a medicament for antagonizing interleukin-8.

Description

Screening method and application of pulegone serving as interleukin-8 antagonist Technical Field The invention relates to the technical field of biological medicines, in particular to a screening method and application of pulegone serving as an interleukin-8 antagonist. Background Interleukin-8 (IL-8), also known as chemokine CXCL8, is a CXC subfamily of 72 amino acid residues having a molecular weight of about 8kDa. IL-8 is synthesized and secreted by endothelial cells, epithelial cells, fibroblasts, monocytes, macrophages and other cells under the stimulation of inflammation, has lower expression level under normal physiological state, and is rapidly up-regulated under the inflammatory conditions such as infection or injury. IL-8, through binding to its specific receptor CXCR1/CXCR2, activates downstream signaling pathways, playing a central role in chemotaxis, activation and tissue infiltration of neutrophils, participating in body defense responses and tissue repair following injury. However, abnormally high expression of IL-8 is closely related to the pathological course of a variety of inflammatory and autoimmune diseases. For example, in synovial tissue and synovial fluid of rheumatoid arthritis patients, IL-8 levels are significantly elevated, which promotes local infiltration of neutrophils into the joint and release of matrix metalloproteinases, reactive oxygen species, etc., exacerbating cartilage and bone destruction. Also, in psoriatic lesions, up-regulation of IL-8 can mediate migration of neutrophils to the epidermis, leading to keratinocyte hyperproliferation and maintenance of a chronic inflammatory state. Currently, inhibition of the IL-8 signaling pathway has become an important strategy for the treatment of related inflammatory diseases. Biological agents, represented by monoclonal antibodies, can inhibit the recruitment and activation of inflammatory cells such as neutrophils by blocking the interaction of IL-8 with CXCR1/CXCR2 receptors, thereby alleviating the disease. However, the existing antibody drug still has the problems of strong immunogenicity, easy induction of anti-idiotype antibodies, reduced drug effect and the like, and adverse reactions such as injection site reaction, fever, cytokine release syndrome and the like may be accompanied in the clinical application process. Therefore, there remains a need in the art to develop a novel inhibitor that is safer, less immunogenic, and effective in blocking the IL-8/CXCR1/2 signaling pathway, to overcome the deficiencies of the prior art, providing a better choice for the treatment of inflammatory and autoimmune diseases. Disclosure of Invention Based on this, it is necessary to provide a screening method and application of pulegone as interleukin-8 antagonist. In one aspect, an embodiment of the present invention provides a method for screening an interleukin-8 antagonist, comprising the steps of: s1, customizing a drug molecule library, and customizing interleukin-8 as a target for later use; S2, performing high-flux molecular screening in a drug molecular library by using a target, and screening drug molecules capable of combining with interleukin-8 targets from the drug molecular library; s3, verifying the in-vitro antagonistic activity of the medicine molecules screened in the step S2, thereby detecting the antagonistic effect of the specific medicine molecules on the target effect, and evaluating the antagonistic effect of the specific medicine molecules on interleukin-8. Preferably, the drug molecular library is an FDA approved 3067 compound drug library. Preferably, in the step S1, the compound drugs in the drug molecule library are dissolved and stored in DMSO with the concentration of 1 mg/mL, the purity of the interleukin-8 target is more than 95%, and the interleukin-8 target is subjected to gradient concentration dilution by deionized water, wherein the amino acid sequence of the interleukin-8 target is shown as SEQ ID NO. 1. Preferably, in step S2, a surface plasmon resonance imaging technique is used for high-throughput screening, which specifically includes: S21, fixing and assembling the drug molecule library into a microfluidic chip; S22, mounting the microfluidic chip in a PlexArray HT SPRi microarray analyzer; s23, sequentially sampling the interleukin-8 target to be tested according to a concentration gradient; s24, analyzing the collected signal data of the detection binding signals through data analysis software Data Processor Stand Alone, sorting the molecular affinity of the analysis results, and screening out the compound drug bound with the target. Preferably, before step S23, a positive control sample is used for sample injection detection in advance, and the quality of the chip is verified according to whether a positive response signal of the positive sample is detected. Preferably, the positive control sample is rapamycin. Preferably, in step S3, the antagonism of interleukin-8 by the compound drug i