A human immunoglobulin e antagonist, screening method and use

Abstract

The present application relates to the technical field of pharmaceutical chemistry, in particular to a human immunoglobulin E antagonist, a screening method and application. The screening method comprises the following steps: taking IgE as a target, screening drug molecules capable of binding with IgE in a high-throughput FDA drug library; testing the antagonizing efficiency of the drug molecules screened in vitro, and screening molecules capable of antagonizing IgE according to the expected antagonizing efficiency. The present application takes IgE as a target, screens drug molecules capable of binding with IgE in a high-throughput FDA drug library, and verifies the antagonizing efficiency of the drug molecules at the molecular level in vitro, so that a molecule capable of antagonizing IgE is screened, and the antagonizing efficiency of the screened thioctic acid is significantly higher than that of common antagonists.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of pharmaceutical chemistry, and particularly relates to a human immunoglobulin E antagonist, a screening method and application. BACKGROUND

[0002] Human immunoglobulin E (IgE) is an important immunoglobulin produced by B lymphocytes transformed into plasma cells under antigen stimulation, which can specifically bind to the corresponding antigen. IgE has high affinity for eosinophils and mast cells, and is the main antibody causing type I allergic reactions. In normal human blood, the content of IgE is extremely low, but it plays an important role in immune response and inflammatory response.

[0003] Under normal physiological function, IgE mainly acts as a key molecule in mediating allergic reactions, which can recognize and bind to allergens such as pollen, dust mites, animal dander, food, drugs, etc., and then activate immune cells such as mast cells and eosinophils, trigger the release of inflammatory mediators such as histamine, and ultimately lead to allergic reaction symptoms such as skin redness, sneezing or asthma. Under normal conditions, the level of IgE is maintained within a relatively stable range to ensure the reaction to potentially harmful substances without triggering excessive or unnecessary immune responses.

[0004] In allergic diseases, IgE is tightly bound to high-affinity receptors on the surface of mast cells and basophils, triggering changes in downstream signaling pathways, further exacerbating allergic reactions. Therefore, IgE-targeted inhibitors become an important means of treating allergic diseases. Antibody drugs block the binding of IgE to high-affinity receptors on the surface of effector cells, thereby blocking IgE-mediated allergic reactions and relieving allergic symptoms. Currently approved IgE inhibitors for clinical use include omalizumab, ligelizumab, etc.

[0005] However, biological antibody drugs have certain side effects or treatment risks, including acute reactions, serum sickness, tumor lysis syndrome, and cytokine release syndrome, etc. Clinical manifestations include local skin reactions at the injection site, fever, and flu-like syndrome. In addition, the humanization modification of antibody drugs has not completely eliminated immunogenicity, and anti-idiotype antibodies may still be produced, leading to drug failure. Therefore, when using IgE-targeted inhibitors, the efficacy and safety need to be weighed.

[0006] IgE plays a key role in a variety of diseases, but there are many technical shortcomings in the treatment of antibody drugs against IgE. First, antibody drugs can cause side effects, including acute allergic reactions, serum sickness, tumor lysis syndrome and cytokine release syndrome, etc., which are manifested as injection site reactions, fever and flu-like symptoms. Second, although the antibody drug has been humanized, its CDR region still has strong immunogenicity, which can produce anti-idiotype antibodies, leading to drug failure. In addition, the structure of the antibody drug is unstable and easily affected by environmental factors, which can occur at any stage of production. Finally, the pharmacokinetic properties of the antibody drug are poor, as the volume is large, the ability to penetrate and accumulate tissues is limited, and it is easily affected by the enzyme degradation mechanism in the body, further reducing its pharmacokinetic properties. SUMMARY

[0007] In view of the deficiencies of the prior art, the present application provides a human immunoglobulin E antagonist, a screening method and application.

[0008] The present application provides a human immunoglobulin E antagonist, which is lipoic acid.

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

[0010] (a) Establish FDA-approved drug library: customize a library containing at least 3067 kinds of chemical drugs, each drug is dissolved in DMSO at a concentration of 1 mg / ml and stored;

[0011] (b) Preparation of human immunoglobulin E target: customize human immunoglobulin E protein target with a purity of at least 95%, and gradient dilute it 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: assemble the FDA drug library into a microfluidic chip and install it into a suitable high-throughput screening analyzer, after verifying the quality of the chip using a positive control sample, sequentially sample the human immunoglobulin E target according to the concentration gradient, detect the interaction with the human immunoglobulin E target, analyze the collected signal data using data analysis software, and sort the results according to the molecular affinity;

[0013] (d) Candidate drug screening: among the results obtained in step (c), identify chemical drugs with significant affinity reduction effect on human immunoglobulin E target, wherein lipoic acid is screened as a candidate drug with potential antagonistic effect;

[0014] (e) Drug molecule level verification: using human immunoglobulin E detection kit, taking known human immunoglobulin E monoclonal drug as positive control, evaluating the antagonistic effect of lipoic acid on human immunoglobulin E, and verifying the antagonistic activity of lipoic acid by calculating the antagonistic rate.

[0015] In further preferred embodiments, the lipoic acid is confirmed to have a significant affinity reduction effect in step (d), and shows an antagonistic rate at least equivalent to that of the positive control drug in step (e) by ELISA detection.

[0016] In further preferred embodiments, the FDA-approved drug library can include more kinds of chemical drugs to improve the diversity and accuracy of screening.

[0017] In further preferred embodiments, the purity of the human immunoglobulin E target can be determined by methods such as SDS-PAGE to ensure that it meets the screening requirements.

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

[0019] In the drug molecule level verification step, the antagonistic rate is calculated as follows: antagonistic rate = 100% - (detection concentration / initial concentration) x 100%.

[0020] In further preferred embodiments, the human immunoglobulin E is as shown in SEQ ID NO: 1.

[0021] The present application screens for a drug molecule that can bind to IgE in the FDA drug library by taking IgE as a target, and verifies the antagonistic efficiency of the screened molecule at the molecular level in vitro, thereby screening for a molecule that can antagonize IgE, and the antagonistic efficiency of the screened lipoic acid is significantly higher than that of ordinary antagonists.

[0022] Another aspect of the present application provides a use of lipoic acid as an antagonist of human immunoglobulin E.

[0023] Another aspect of the present application provides an antiallergic pharmaceutical composition containing lipoic acid.

[0024] Another aspect of the present application provides a method for treating or preventing allergic diseases using lipoic acid. BRIEF DESCRIPTION OF DRAWINGS

[0025] The above and other objects, features and advantages of the present application will become more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: The same reference numerals are intended to denote the same components throughout the specification. The drawings are not intended to be drawn to scale. The emphasis is placed rather on illustrating the principles of the present application.

[0026] Figure 1 Figure 5 is a signal curve of lipoic acid collected by injection when the IgE protein concentration is 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM, respectively.

[0027] Figure 2 Figure 6 is a molecular structure diagram of lipoic acid.

[0028] Figure 3 Figure 7 is a dose-effect analysis diagram of omalizumab.

[0029] Figure 4 Figure 8 is a dose-effect analysis diagram of S3996 lipoic acid. DETAILED DESCRIPTION

[0030] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0032] The screening method of the immunoglobulin E antagonist of the present application comprises the following steps:

[0033] First, IgE is taken as a target to find drug molecules that can bind to IgE in a high-throughput screening FDA drug library; then, the drug molecules are tested for antagonistic efficiency in vivo to screen out molecules that can effectively antagonize IgE. In the specific process, 3067 kinds of FDA-approved chemical drugs and IgE protein targets with determined purity are used for screening by microfluidic chips and PlexArray HT SPRi microarray analyzers, and finally small molecule compounds meeting the requirements, i.e., lipoic acid, are screened out by data analysis software. Then, the antagonistic effect of lipoic acid is detected by an immunoglobulin E ELISA detection kit and evaluated, and the IgE monoclonal antibody drug adalimumab is also used as a positive control in the evaluation process. The whole screening process includes the steps of incubation of IgE and lipoic acid, immune reaction and color development, determination of absorbance and calculation of concentration.

[0034] More specifically, the method comprises the following steps

[0035] (a) FDA drug library establishment. 3067 kinds of FDA-approved chemical drugs are customized and stored in DMSO at 1 mg / ml.

[0036] (b) IgE target preparation. Custom IgE protein target with 95% purity determined by SDS-PAGE. Dilute IgE gradient with deionized water to 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM.

[0037] (c) Molecular screening with IgE as target. Assemble FDA drug library chip into microfluidic chip and install into PlexArray HT SPRi microarray analyzer, detect with positive sample rapamycin to verify chip quality. Inject test protein in order of concentration gradient, finally analyze collected signal data with data analysis software Data Processor Stand Alone, and rank results by molecular affinity.

[0038] (d) Drug molecule level verification. Use human (Human) immunoglobulin E (IgE) ELISA detection kit (Shenzhen Zike) to detect the antagonistic effect of lipoic acid on IgE, and use IgE monoclonal drug omalizumab as a positive control. Detect absorbance with a microplate reader, and calculate antagonistic rate according to detection concentration, antagonistic rate = 100% - (detection concentration / initial concentration).

[0039] The IgE amino acid sequence is shown in SEQ ID NO: 1.

[0040] SEQ ID NO: 1

[0041] 1: CADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTR

[0042] 61: KEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYA

[0043] 121: FATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFS

[0044] 181: RLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK

[0045] In a preferred embodiment, the high-throughput screening step of the FDA drug library chip with IgE as the target is as follows: (1) Take out the prepared FDA drug library chip, block it in 1% BSA solution for 2h, wash the chip with pure water and dry it, and then assemble it into a microfluidic chip.

[0046] (2) 1 mg / ml human immunoglobulin E (IgE) protein was diluted with PBST buffer.

[0047] (3) The microfluidic chip was installed into the PlexArray HT SPRi microarray analyzer, and FKBP12 positive sample with a concentration of 100 nM was first flowed through 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.

[0048] (4) PBST buffer was flushed at a flow rate of 1 μl / s for 300 sec, and the test protein was injected in gradient concentrations in sequence at a flow rate of 1 μl / sec for 300 sec. PBST buffer was dissociated at a flow rate of 1 μl / s for 300 sec. The above steps were repeated until all concentrations of protein were injected, and the signal data were collected.

[0049] (5) The chip was stored after regeneration by using Gly-HCl buffer (pH = 2.0) at a flow rate of 1 μl / s for 120 s of binding and 180 s of dissociation.

[0050] (6) The collected signal data were analyzed by using the data analysis software Data Processor Stand Alone, and the results were ranked according to the molecular affinity.

[0051] Results refer to Figure 1 and Figure 2 , and the small molecule compound lipoic acid with the number S3996 was screened. It can be seen that the stronger the detected binding signal is, the higher the protein concentration is. Among all the small molecules, the affinity ranks third, and it is used as a candidate molecule for subsequent research.

[0052] In the preferred embodiment, the dose-effect analysis of the positive control omalizumab antagonizing IgE is as follows: (1) 50 μl of 10 μg / ml IgE protein was added to an EP tube, and different concentrations of omalizumab were added, and incubated at 37°C for 1 h.

[0053] (2) 100 μl of protein small molecule mixture or standard was sequentially added to the coated microwells coated with IgE antibody, and 100 μl of HRP-labeled IgE antibody was added, and incubated at 37°C for 1 h, and then the liquid was discarded, and washed 5 times, and TMB was developed.

[0054] (3) The absorbance at 450 nm was measured by using an enzyme-labeled instrument, a standard curve was established, and the detection concentration was calculated. The antagonistic rate was calculated according to the detection concentration, and the antagonistic rate = 100%-(detection concentration / initial concentration).

[0055] Please refer to Figure 3And Table 1, the results were analyzed: the dose-effect analysis results of omalizumab found that the antagonistic effect reached 22.96% at a concentration of 120 μg / ml.

[0056] Table 1:

[0057]

[0058] In a preferred embodiment, the dose-effect analysis step of lipoic acid against IgE is as follows:

[0059] (1) Add 50 μl of 10 μg / ml IgE protein to an EP tube, add lipoic acid of different concentrations respectively, and incubate at 37°C for 1 h.

[0060] (2) Add 100 μl of protein small molecule mixture or standard to the coated micro-well coated with IgE antibody in turn, and add 100 μl of HRP-labeled IgE antibody, incubate at 37°C for 1 h, then discard the liquid, wash 5 times, and develop with TMB.

[0061] (3) Measure the absorbance at 450 nm with an enzyme-labeled instrument, establish a standard curve, and calculate the detection concentration. According to the detection concentration, calculate the antagonistic rate, and the antagonistic rate = 100%-(detection concentration / initial concentration).

[0062] Please refer to Figure 4 And Table 2, the dose-effect analysis results of S3996 lipoic acid found that the concentration of the equivalent antagonistic effect of the antagonist was 7.28 μg / ml.

[0063] Table 2:

[0064]

[0065] Among the above materials and reagents:

[0066] The FDA drug library chip was purchased from Selleck Chemicals, human immunoglobulin E was purchased from Boster Biological Technology Co., Ltd., PBST buffer was purchased from Thermo Fisher Scientific, Gly-HCl buffer was purchased from Thermo Fisher Scientific, omalizumab was purchased from MedChemexpress Biological Technology Co., Ltd. of the United States, and lipoic acid was purchased from MedChemexpress Biological Technology Co., Ltd. of the United States.

[0067] In summary, the present application screens the drug molecules that can bind to IgE in the FDA drug library by taking IgE as the target, and verifies the antagonistic efficiency at the molecular level in vitro, screens a molecule that can antagonize IgE, and the antagonistic efficiency of the screened lipoic acid is significantly higher than that of the ordinary antagonists. First, the screened drug lipoic acid of the present application effectively avoids the side effects such as acute allergic reaction, and ensures the safety of the patient. Secondly, by optimizing the structure of the drug, the present application reduces the immunogenicity of the CDR region, significantly reduces the generation of anti-idiotype antibodies, and prolongs the effective period of the drug. Thirdly, lipoic acid is stable in structure and is not easily affected by environmental factors. Finally, lipoic acid performs well in pharmacokinetics, has a small volume and stronger tissue penetration ability, and can resist enzymatic degradation in the body, thereby improving the bioavailability and efficacy of the drug. These advantages make the present application have broad application prospects in the treatment of IgE-related diseases.

[0068] Lipoic acid (LA), 5-[3-(1,2-dithia cyclo)]pentanoic acid, was first isolated from bovine liver by Reed et al. in 1951, and is a trace element with multiple biological and pharmacological properties. It is a limiting essential nutrient required for cells to utilize energy sources such as sugars, and is the strongest natural antioxidant in nature. Due to the alkyl chain and carboxyl end of lipoic acid, it has both fat-soluble and water-soluble properties, can smoothly pass through the blood-brain barrier into any tissue and has almost no toxic side effects. In normal physiological functions, lipoic acid participates in the tricarboxylic acid cycle as an important coenzyme, and as an important repair factor in biological systems, removes oxygen free radicals and active oxygen, and reduces the attack of free radicals on the body. In addition to a small amount of lipoic acid synthesized by fatty acids and cysteine in the body, the main lipoic acid in the human body is obtained from food intake, and the lipoic acid entering the body can be rapidly absorbed into the blood and accumulated in various tissues such as the heart, liver, skeletal muscle, etc. Studies have found that lipoic acid has been used to treat various chronic diseases such as diabetes, liver fibrosis and neurodegenerative diseases, and in vitro supplementation of lipoic acid can inhibit lipid oxidation in neural tissue, prevent protein glycosylation, inhibit aldose reductase, and prevent glucose or galactose from being converted into sorbitol. It is commonly used in clinical treatment and relief of peripheral neuropathy caused by late-stage diabetes; it can also strengthen the function of liver activity, improve energy metabolism rate, synergistically reduce free radical damage to liver cells with other antioxidants, reduce inflammation, maintain liver function, clear fat accumulation caused by non-alcoholic fatty liver, and reduce the burden on the liver. In addition, lipoic acid is a super strong antioxidant that can preserve and regenerate other antioxidants, balance blood glucose concentration, enhance the body's immune system, protect against free radical damage, participate in energy metabolism, increase the ability of other antioxidants to eliminate free radicals, and promote the recovery of insulin sensitivity.

[0069] In addition, lipoic acid has significant anti-inflammatory effects in inflammatory reactions, and its antioxidant capacity plays an important role in the anti-inflammatory process. Studies have shown that lipoic acid can prevent the expression of ICAM-1 and the adhesion of vascular endothelial human mononuclear cells, and inhibit the expression of NF-κB-dependent metalloproteinase-9, while also reducing the expression of other inflammatory factors such as IL-6 and TNF-α. In summary, lipoic acid is a vitamin-like disulfide compound with rich biological activity and great application prospects.

[0070] In another aspect, the present application relates to a new use of lipoic acid, specifically as an antagonist of human immunoglobulin E (IgE). Lipoic acid, through its unique chemical structure and biological activity, can effectively bind to IgE, thereby inhibiting IgE-mediated immune responses and reducing the occurrence of allergic reactions.

[0071] In another aspect, the present application provides an anti-allergic pharmaceutical composition containing lipoic acid, which also includes a pharmaceutically acceptable carrier or excipient. This composition, through the antagonistic effect of lipoic acid on IgE, can effectively alleviate or prevent allergic reactions, including but not limited to hay fever, asthma, and food allergies, etc.

[0072] In another aspect, the present application provides a method for preparing an anti-allergic pharmaceutical composition containing lipoic acid, which includes mixing lipoic acid with a pharmaceutically acceptable carrier or excipient.

[0073] In another aspect, the present application provides a method for treating or preventing allergic diseases using lipoic acid, which includes administering an effective amount of lipoic acid or its pharmaceutical composition to a patient. This method, by inhibiting IgE-mediated immune responses, can effectively alleviate or prevent the symptoms of allergic diseases and improve the quality of life of patients.

[0074] In another aspect, the present application also relates to the use of lipoic acid in the preparation of a medicament for regulating the human immune system. Lipoic acid, through its antioxidant, anti-inflammatory, and immunomodulatory properties, can regulate the function of the immune system, reduce the production and activity of IgE, and thus help maintain the balance and stability of the immune system.

[0075] In another aspect, the present application provides an immunomodulator containing lipoic acid, which also includes other immunomodulatory components and / or pharmaceutically acceptable carriers or excipients. This immunomodulator, through the synergistic effect of lipoic acid and other components, can effectively regulate the function of the immune system, reduce IgE-mediated immune responses, and thus prevent or alleviate the occurrence of allergic diseases.

[0076] Although the embodiments of the present application have been shown and described above, it is understood that the above embodiments are exemplary, and are not to be interpreted as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A method for screening human immunoglobulin E antagonists, characterized in that, The antagonist is lipoic acid, and the human immunoglobulin E antagonist was obtained through the following screening method: (a) Establish an FDA-approved drug library: Customize a library containing at least 3,067 compound drugs, each dissolved in DMSO at a concentration of 1 mg / ml and stored. (b) Preparation of human immunoglobulin E target: Customize human immunoglobulin E protein target with a purity of at least 95% and serially dilute it to at least five concentration levels, said five concentrations including 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM; wherein, human immunoglobulin E is as shown in SEQ ID NO: 1; (c) Molecular screening: The FDA drug library is assembled into a microfluidic chip and installed in a suitable high-throughput screening analyzer. After verifying the chip quality using positive control samples, human immunoglobulin E targets are injected sequentially according to the concentration gradient. The interaction with human immunoglobulin E targets is detected, and the collected signal data is analyzed using data analysis software. The results are sorted according to the molecular affinity. (d) Candidate drug screening: In the results obtained in step (c), compound drugs that have a significant affinity-reducing effect on the human immunoglobulin E target were identified, among which lipoic acid was screened as a candidate drug with potential antagonistic effect; (e) Drug molecular level validation: Using a human immunoglobulin E detection kit and a known human immunoglobulin E monoclonal antibody drug as a positive control, the antagonistic effect of lipoic acid on human immunoglobulin E was evaluated, and the antagonistic activity of lipoic acid was verified by calculating the antagonistic rate.

2. The screening method for human immunoglobulin E antagonists according to claim 1, characterized in that: The lipoic acid was confirmed to have a significant affinity-reducing effect in step (d) and showed an antagonistic rate at least comparable to that of the positive control drug by ELISA detection in step (e).

3. The method for screening human immunoglobulin E antagonists according to claim 2, characterized in that: The FDA-approved drug library can contain a wider variety of compound drugs to improve the diversity and accuracy of screening.

4. The method for screening human immunoglobulin E antagonists according to claim 3, characterized in that: The purity of the human immunoglobulin E target can be determined by SDS-PAGE to ensure that it meets the screening requirements.

5. The method for screening human immunoglobulin E antagonists according to claim 4, characterized in that: The high-throughput screening analyzer can be a PlexArray HT SPRi microarray analyzer or other instruments suitable for molecular screening; In the drug molecule-level validation step, the antagonism rate is calculated as follows: Antagonism rate = 100% - (detected concentration / initial concentration) × 100%.

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