Interleukin 5 receptor antagonist, screening method and application

Abstract

The present invention relates to the technical field of pharmaceutical chemistry, and an interleukin 5 receptor antagonist, a screening method, and an application screening method include the following steps: taking the interleukin 5 receptor as a target, and screening the drug molecules that can bind to the interleukin 5 receptor in the FDA drug library in high throughput; testing the antagonistic efficiency of the above-screened drug molecules in vivo, and screening out molecules that can antagonize the interleukin 5 receptor according to the expected antagonistic efficiency. The present invention uses the interleukin 5 receptor as a target, and high-throughput screening of drug molecules that can bind to the interleukin 5 receptor in the FDA drug library, and verifies its antagonistic efficiency at the in vitro molecular level, and screens out a molecule that can antagonize the interleukin 5 receptor, and the screened methyl hesperidin antagonistic efficiency has a significant antagonistic efficiency compared to ordinary antagonists. Methyl hesperidin is an important natural flavonoid compound that can be used as an antagonist of IL-5R.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry and relates to an interleukin 5 receptor antagonist, a screening method and an application thereof. Background Art

[0002] Interleukin-5 (IL-5) is a multifunctional cytokine that is mainly produced by activated T cells, mast cells, Th2 cells and other immune system cells, and plays a key role in immune regulation and inflammatory response. IL-5 can promote the growth, differentiation, recruitment, activation and survival of eosinophils and basophils, thereby enhancing the body's immune response to parasitic infections and allergic diseases. In addition, it can stimulate B cell proliferation and increase antibody secretion, regulate hematopoiesis, and promote inflammatory response.

[0003] IL-5 exerts its biological effects by binding to the IL-5 receptor (IL-5R) on the surface of target cells. IL-5R is composed of α chain and β chain, triggering downstream signal transduction pathways to regulate biological processes such as cell proliferation, differentiation, and apoptosis. Given the important role of IL-5 in immune response and inflammatory response, it has broad application prospects in the medical field, especially in the treatment of allergic diseases.

[0004] Drugs targeting IL-5 or IL-5R can inhibit the activity of eosinophils, reduce allergic reactions and inflammation. Biological agents mainly composed of IL-5R targeted inhibitors are an important component of IL-5R antagonists. Antibody drugs inhibit the accumulation of immune cells and inflammatory factors by blocking the interaction between IL-5 and IL-5R, thereby alleviating disease symptoms. These biological agents can replace or combine with hormone therapy to reduce the incidence of adverse drug reactions.

[0005] However, biologic antibody drugs also have certain side effects or treatment risks, such as acute reactions after injection, including acute allergic 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. Although antibody drugs have been humanized, immunogenicity has not been completely eliminated, and anti-idiotypic antibodies may still be produced, causing drug failure. Therefore, when using these biologics, it is necessary to weigh their efficacy and safety.

[0006] IL-5R plays a key role in many diseases, but there are technical disadvantages in antibody drug therapy targeting IL-5R. First, antibody drugs may cause side effects, such as acute allergic reactions, serum sickness, tumor lysis syndrome, and cytokine release syndrome, which are clinically manifested as local skin reactions, fever, and flu-like syndrome. Secondly, although antibody drugs have been humanized, immunogenicity has not been completely eliminated, which may lead to drug failure. In addition, antibody drugs are structurally unstable and susceptible to environmental physical stress, and structural changes may occur at any stage from protein expression to processing to storage. Finally, antibody drugs have poor pharmacokinetic properties, large size, limited ability to penetrate and accumulate tissues, and are susceptible to enzymatic degradation mechanisms in the body, further reducing their pharmacokinetic properties. Summary of the invention

[0007] In view of the deficiencies of the prior art, the present invention provides a method for screening an interleukin 5 receptor antagonist and the use of methyl hesperidin as an antagonist.

[0008] 1. An interleukin 5 receptor antagonist, characterized in that the antagonist is methyl hesperidin.

[0009] In a further preferred embodiment, the methyl hesperidin is obtained by the following screening method:

[0010] (a) Establishment of an FDA-approved drug library: A library containing at least 3067 compound drugs was customized, each drug was dissolved in DMSO at a concentration of 1 mg / ml and stored;

[0011] (b) preparing interleukin 5 receptor targets: customizing interleukin 5 receptor protein targets with a purity of at least 95%, and gradiently diluting the targets into at least five concentration levels, including but not limited to 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM;

[0012] (c) Molecular screening: The FDA drug library is assembled into a microfluidic chip and installed in an appropriate high-throughput screening analyzer. After the chip quality is verified using a positive control sample, the interleukin-5 receptor target is injected in sequence according to a concentration gradient. The interaction with the interleukin-5 receptor target is detected, and the collected signal data is analyzed using data analysis software, and the results are ranked according to the molecular affinity.

[0013] (d) Candidate drug screening: In the results obtained in step (c), a compound drug having a significant affinity reduction effect on the interleukin 5 receptor target is identified, wherein methyl hesperidin is screened as a candidate drug with potential antagonistic effect;

[0014] (e) Drug molecular level verification: An interleukin 5 receptor detection kit was used, with known interleukin 5 receptor monoclonal antibody drugs as positive control, to evaluate the antagonistic effect of methyl hesperidin on interleukin 5 receptor, and the antagonistic activity of methyl hesperidin was verified by calculating the antagonism rate.

[0015] In a further preferred embodiment, the methyl hesperidin is confirmed to have a significant affinity-reducing effect in step (d), and shows an antagonism rate at least equivalent to that of the positive control drug through ELISA detection in step (e).

[0016] In a further preferred embodiment, the FDA-approved drug library may contain more types of compound drugs to improve the diversity and accuracy of screening;

[0017] The purity of the interleukin 5 receptor target can be measured by SDS-PAGE and other methods to ensure that it meets the screening requirements.

[0018] In a further preferred embodiment, the high-throughput screening analyzer may be a PlexArray HT SPRi microarray analyzer or other instruments suitable for molecular screening;

[0019] In the drug molecular level verification step, the antagonism rate is calculated as follows: antagonism rate = 100% - (detection concentration / initial concentration) × 100%;

[0020] In a further preferred embodiment, the interleukin 5 receptor is as shown in SEQ ID NO:1.

[0021] Another aspect of the present invention provides a pharmaceutical composition, which comprises the methyl hesperidin as an active ingredient and one or more pharmaceutically acceptable carriers or excipients.

[0022] In a further preferred embodiment, the pharmaceutical composition further comprises one or more other therapeutic agents for synergistically enhancing the antagonistic effect on IL-5R.

[0023] Another aspect of the present invention provides a use of methyl hesperidin in the preparation of a medicament for treating or preventing diseases associated with IL-5R, wherein the diseases include but are not limited to asthma, eosinophilia and allergic inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and other purposes, features and advantages of the present invention will become more apparent by more specific description of the preferred embodiments of the present invention shown in the accompanying drawings. The same reference numerals indicate the same parts in all the accompanying drawings, and the accompanying drawings are not deliberately scaled to the actual size, but the focus is on illustrating the main purpose of the present invention.

[0025] Figure 1 The methyl hesperidin signal curves collected when the IL-5R protein concentrations were 500.0 nM, 250.0 nM, 125.0 nM, 62.5 nM and 31.2 nM, respectively.

[0026] Figure 2 This is the molecular structure of methyl hesperidin.

[0027] Figure 3 This is a dose-effect analysis diagram of the positive control benralizumab at concentrations of 0, 3, 6, 12, 24, 48, and 96 μg / ml.

[0028] Figure 4 This is a dose-effect analysis diagram for methyl hesperidin of 0, 1.19, 4.74, 18.97, 75.9, 303.6, and 1214.39 μg / ml. DETAILED DESCRIPTION

[0029] To facilitate understanding of the present invention, a more comprehensive description of the present invention will be given below with reference to the relevant drawings.

[0030] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art. The terms used in the specification herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0031] The present invention provides a method for screening methyl hesperidin as an interleukin 5 receptor antagonist, comprising the following steps:

[0032] First, an FDA drug library was established, containing 3067 chemical drugs; secondly, the TNF-α target, namely the IL-5R protein, was prepared and gradiently diluted. Then, IL-5R was used as the target, and molecular screening was performed using a microfluidic chip. After verifying the chip quality, the data was analyzed. Finally, the antagonistic effect of the screened drug molecules (such as methyl hesperidin) was verified by the human IL-5R ELISA detection kit, and the antagonistic rate was calculated using benralizumab as a positive control.

[0033] More specifically, the steps include:

[0034] 1. Establishment of FDA drug library: 3067 FDA-approved compound drugs were customized and stored in 1mg / ml DMSO.

[0035] 2. Preparation of TNF-α target. The IL-5R protein target was customized, and its purity was determined to be 95% by SDS-PAGE. IL-5R was gradiently diluted with deionized water to 500nM, 250nM, 125nM, 62.5nM and 31.25nM.

[0036] 3. Molecular screening with IL-5R as the target. The FDA drug library chip was assembled into a microfluidic chip and installed in the PlexArray HT SPRi microarray analyzer. The positive sample rapamycin was tested to verify the chip quality. The proteins to be tested were injected in sequence according to the concentration gradient. Finally, the collected signal data was analyzed using the data analysis software Data Processor Stand Alone, and the results were sorted by molecular affinity.

[0037] 4. Drug molecular level verification. The human interleukin 5 receptor (IL-5R) ELISA test kit (Shenzhen Zike) was used to detect the antagonistic effect of methyl hesperidin on IL-5R, and the IL-5R monoclonal antibody drug benralizumab was used as a positive control. The absorbance was detected by an enzyme reader, and the antagonism rate was calculated based on the detection concentration, antagonism rate = 100% - (detection concentration / initial concentration).

[0038] SEQ ID NO:1

[0039] 1:DLLPDEKISLLPPVNFTIKVTGLAQVLLQWKPNPDQEQRNVNLEYQVKINAPKEDDYETR

[0040] 61:ITESKCVTILHKGFSASVRTILQNDHSLLASSWASAELHAPPGSPGTSIVNLTCTTNTTE

[0041] 121:DNYSRLRSYQVSLHCTWLVGTDAPEDTQYFLYYRYGSWTEECQEYSKDTTLGRNIACWFPR

[0042] 181:TFILSKGRDWLAVLVNGSSKHSAIRPFDQLFALHAIDQINPPLNVTAEIEGTRLSIQWEK

[0043] 241:PVSAFPIHCFDYEVKIHNTRNGYLQIEKLMTNAFISIIDDLSKYDVQVRAAVSSMCREAG

[0044] 301:LWSEWSQPIYVGNDE

[0045] In a preferred embodiment, the steps of high-throughput screening of the FDA drug library chip using IL-5R as a target are as follows:

[0046] (1) The prepared FDA drug library chip was taken out and placed in a 1% BSA solution for blocking for 2 h. The chip was washed with pure water and dried before being assembled into a microfluidic chip.

[0047] (2) Dilute the 1 mg / ml interleukin 5 receptor (IL-5R) protein in PBST buffer in multiple ratios.

[0048] (3) The microfluidic chip was installed in the PlexArray HT SPRi microarray analyzer, and a 100 nM FKBP12 positive sample was first circulated at a flow rate of 1 μl / sec for 300 sec. The positive response signal of rapamycin was detected to verify the quality of the chip.

[0049] (4) The cells were washed with PBST buffer at a flow rate of 1 μl / s for 300 sec, and the proteins to be tested were injected in gradient concentrations at a flow rate of 1 μl / sec for 300 sec. The cells were dissociated with PBST buffer at a flow rate of 1 μl / s for 300 sec. The above steps were repeated until all concentrations of proteins were injected and the signal data were collected.

[0050] (5) The chip was regenerated with Gly-HCl buffer (pH=2.0) at a flow rate of 1 μl / s, with binding for 120 s and dissociation for 180 s, and then stored.

[0051] (6) Use the data analysis software Data Processor Stand Alone to analyze the collected signal data and rank the results according to the molecular affinity.

[0052] Results reference Figure 1 and Figure 2 , and screened out the small molecule compound methylhesperidine numbered S2323. The results show that as the protein concentration increases, the detected binding signal becomes stronger, and its affinity ranks fourth among all small molecules. It is used as a candidate molecule for subsequent research.

[0053] In a preferred embodiment, the steps for analyzing the efficacy of the positive control benralizumab in antagonizing IL-5R are as follows:

[0054] (1) Add 50 μl of 100 pg / ml IL-5R protein to an EP tube, add different concentrations of benralizumab, and incubate at 37°C for 1 h.

[0055] (2) Add 100 μl of protein small molecule mixture or standard and 100 μl of HRP-labeled IL-5R antibody to the coated microwells pre-coated with IL-5R antibody, in turn, incubate at 37°C for 1 h, discard the solution, wash 5 times, and develop color with TMB.

[0056] (3) Use an enzyme-labeled instrument to measure the absorbance at 450 nm and calculate the detection concentration after establishing a standard curve.

[0057] The antagonism rate was calculated according to the test concentration, antagonism rate = 100% - (test concentration / initial concentration).

[0058] Please refer to Figure 3 As shown in Table 1, the results of the dose-effect analysis of subcutaneous injection of 30 mg of benralizumab showed that the antagonistic effect reached 16.93% when the monoclonal antibody concentration was 6.00 μg / ml (working concentration).

[0059] Table 1:

[0060]

[0061] In a preferred embodiment, the steps of analyzing the efficacy of lipoic acid against IgE are as follows:

[0062] (1) Add 50 μl of 120 pg / ml IL-5R protein to an EP tube, add different concentrations of methyl hesperidin, and incubate at 37°C for 1 h.

[0063] (2) Add 100 μl of protein small molecule mixture or standard and 100 μl of HRP-labeled IL-5R antibody to the coated microwells pre-coated with IL-5R antibody, in turn, incubate at 37°C for 1 h, discard the solution, wash 5 times, and develop color with TMB.

[0064] (3) Use an enzyme-labeled instrument to measure the absorbance at 450 nm and calculate the detection concentration after establishing a standard curve.

[0065] The antagonism rate was calculated according to the test concentration, antagonism rate = 100% - (test concentration / initial concentration).

[0066] Results reference Figure 4 As shown in Table 2, the results of the dose-effect analysis of S2323 methyl hesperidin showed that the concentration of the antagonist with the same antagonistic effect was 2.50 pg / ml.

[0067] Table 2:

[0068]

[0069] The above materials and reagents include:

[0070] FDA drug library chip was purchased from Selleck Chemicals, interleukin 5 receptor protein was purchased from Biopsys Biotech, PBST buffer was purchased from Thermo Fisher Scientific, Gly-HCl buffer was purchased from Thermo Fisher Scientific, benralizumab was purchased from MedChemexpress Biotech, and human interleukin 5 receptor (IL-5R) ELISA detection kit was purchased from Shenzhen Zike Biotechnology Co., Ltd.

[0071] In summary, the present invention uses IL-5R as a target, high-throughput screening of drug molecules that can bind to IL-5R in the FDA drug library, and verifies its antagonistic efficiency at the in vitro molecular level, thereby screening out a molecule that can antagonize IL-5R. The methyl hesperidin of the present invention has significant safety and can greatly reduce the risk of side effects such as acute allergic reactions, serum sickness, tumor lysis syndrome and cytokine release syndrome. The clinical manifestations are very few local skin reactions, fever and flu-like syndrome, providing patients with a more comfortable and safe treatment experience. The methyl hesperidin of the present invention has extremely low immunogenicity through advanced biotechnology and humanization transformation, effectively avoiding the problem of drug failure caused by immune response. At the same time, the drug structure is stable and can resist the influence of environmental physical stress. The entire process from protein expression to processing to storage maintains the integrity of the structure, ensuring the efficacy and safety of the drug, and can maintain its biological activity under various environmental conditions. This means that the drug is not prone to denaturation or degradation during storage and transportation, thereby improving the availability and reliability of the drug. The methyl hesperidin of the present invention has excellent pharmacokinetic properties, is small in size, has strong ability to penetrate and accumulate tissues, can quickly reach the lesion site and exert a therapeutic effect. At the same time, the drug has a long half-life in the body and is not easily affected by the enzyme degradation mechanism, thereby ensuring the sustained efficacy and stability of the drug.

[0072] Methylhesperidine is an important natural flavonoid compound, widely found in the peel, seeds and pulp of citrus fruits, with multiple pharmacological effects and potential clinical applications. The chemical formula of methylhesperidine is 3'-methyl-7-(rhamnose-L-glucose) hesperidin, which has the same efficacy as vitamin P, can enhance the effect of vitamin C, and has strong antiviral and antibacterial effects, and can interfere with the viral replication cycle or bacterial growth metabolism. At the same time, it can relieve tissue damage and discomfort caused by inflammation by regulating the activity of immune cells and inhibiting the release of inflammatory mediators, and can also enhance the elasticity and toughness of blood vessel walls, prevent and treat capillary bleeding, gingival bleeding, etc. In addition, as a natural antioxidant, methylhesperidin can scavenge free radicals in the body and reduce the damage of oxidative stress to cells. It also has the same pharmacological effects as hesperidin, such as anti-inflammatory, antioxidant, antibacterial, antiviral, anti-diabetic complications, and beneficial to the cardiovascular system. Among them, studies have pointed out that 100 mg / kg of hesperidin can reduce the neuropathological changes and the expression of proinflammatory factors TNF-α and IL-1β in rats with spinal cord injury, possibly through the inhibition of the nuclear factor E2 related factor 2 (Nrf2) / heme oxygenase-1 pathway. On the other hand, the present invention provides a pharmaceutical composition, which comprises the methyl hesperidin as an active ingredient and one or more pharmaceutically acceptable carriers or excipients.

[0073] Another aspect of the present invention provides a pharmaceutical composition, which comprises the methyl hesperidin as an active ingredient and one or more pharmaceutically acceptable carriers or excipients.

[0074] In a further preferred embodiment, the pharmaceutical composition further comprises one or more other therapeutic agents for synergistically enhancing the antagonistic effect on IL-5R.

[0075] Another aspect of the present invention provides a use of methyl hesperidin in the preparation of a medicament for treating or preventing diseases associated with IL-5R, wherein the diseases include but are not limited to asthma, eosinophilia and allergic inflammation.

[0076] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in the field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. An interleukin-5 receptor antagonist, characterized in that The antagonist is methyl hesperidin.

2. The interleukin 5 receptor antagonist according to claim 1, characterized in that The methyl hesperidin is obtained by the following screening method: (a) Establishment of an FDA-approved drug library: A library containing at least 3067 compound drugs was customized, each drug was dissolved in DMSO at a concentration of 1 mg / ml and stored; (b) preparing interleukin 5 receptor targets: customizing interleukin 5 receptor protein targets with a purity of at least 95%, and gradiently diluting the targets into at least five concentration levels, including but not limited to 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM; (c) Molecular screening: The FDA drug library is assembled into a microfluidic chip and installed in an appropriate high-throughput screening analyzer. After the chip quality is verified using a positive control sample, the interleukin-5 receptor target is injected in sequence according to a concentration gradient. The interaction with the interleukin-5 receptor target is detected, and the collected signal data is analyzed using data analysis software, and the results are ranked according to the molecular affinity. (d) Candidate drug screening: In the results obtained in step (c), a compound drug having a significant affinity reduction effect on the interleukin 5 receptor target is identified, wherein methyl hesperidin is screened as a candidate drug with potential antagonistic effect; (e) Drug molecular level verification: An interleukin 5 receptor detection kit was used, with known interleukin 5 receptor monoclonal antibody drugs as positive control, to evaluate the antagonistic effect of methyl hesperidin on interleukin 5 receptor, and the antagonistic activity of methyl hesperidin was verified by calculating the antagonism rate.

3. The interleukin 5 receptor antagonist according to claim 2, characterized in that The methyl hesperidin is confirmed to have a significant affinity-reducing effect in step (d), and shows an antagonism rate at least equivalent to that of the positive control drug through ELISA detection in step (e).

4. The interleukin 5 receptor antagonist according to claim 3, characterized in that: The FDA-approved drug library may contain more types of compound drugs to improve the diversity and accuracy of screening; The purity of the interleukin 5 receptor target can be measured by SDS-PAGE and other methods to ensure that it meets the screening requirements.

5. The interleukin 5 receptor antagonist according to any one of claims 2 to 4, characterized in that: The high-throughput screening analyzer may be a PlexArray HT SPRi microarray analyzer or other instruments suitable for molecular screening; In the drug molecule level verification step, the antagonism rate is calculated as follows: antagonism rate = 100% - (detection concentration / initial concentration) x 100%.

6. The interleukin 5 receptor antagonist according to any one of claims 2 to 4, characterized in that: The interleukin 5 receptor is shown in SEQ ID NO:

1.

7. A pharmaceutical composition, characterized in that The composition comprises the methyl hesperidin according to claim 1 or 2 as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further comprises one or more other therapeutic agents for synergistically enhancing the antagonistic effect on IL-5R.

9. Use of methyl hesperidin according to claim 1 or 2 in the preparation of a medicament for treating or preventing diseases associated with IL-5R, wherein the diseases include but are not limited to asthma, eosinophilia and allergic inflammation.

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