Human immune globulin E antagonist, screening method and application

Abstract

The invention relates to the technical field of medicinal chemistry, in particular to a human immune globulin E antagonist, a screening method and application. The screening method comprises the following steps: by taking IgE as a target spot, screening drug molecules capable of being combined with IgE in an FDA drug library at high throughput; and carrying out an antagonism efficiency test on the screened drug molecules in vivo, and screening the molecules capable of antagonizing IgE according to the expected antagonism efficiency. Drug molecules capable of being combined with IgE in an FDA drug library are screened in a high-throughput mode with the IgE as a target spot, the antagonism efficiency of the drug molecules is verified at the in-vitro molecular level, a molecule capable of antagonizing the IgE is screened out, and the antagonism efficiency of the screened lipoic acid is remarkable compared with that of a common antagonist.

Description

Technical Field

[0001] The present invention relates to the technical field of medicinal chemistry, and in particular to a human immunoglobulin E antagonist, a screening method and an application thereof. Background Art

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

[0003] Under normal physiological functions, IgE mainly acts as a key molecule that mediates allergic reactions. It 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, triggering the release of inflammatory mediators such as histamine, and ultimately leading to allergic reaction symptoms such as skin redness, sneezing or asthma. Under normal conditions, the level of IgE remains within a relatively stable range to ensure that it responds to potentially harmful substances without triggering excessive or unnecessary immune responses.

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

[0005] However, biologic antibody drugs have certain side effects or treatment risks, including acute 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. In addition, the humanization of antibody drugs does not completely eliminate immunogenicity, and anti-idiotypic antibodies may still be produced, causing drug failure. Therefore, when using IgE targeted inhibitors, it is necessary to weigh their efficacy and safety.

[0006] IgE plays a key role in many diseases, but antibody drug treatments targeting IgE have several technical disadvantages. First, antibody drugs may cause side effects, including acute allergic reactions, serum sickness, tumor lysis syndrome, and cytokine release syndrome, which are clinically manifested as injection site reactions, fever, and flu-like symptoms. Secondly, although antibody drugs have been humanized, their CDR regions are still highly immunogenic and may produce anti-idiotypic antibodies, resulting in drug failure. In addition, the structure of antibody drugs is unstable and is easily affected by environmental factors and changes, which may occur at any stage in the production process. Finally, the pharmacokinetic properties of antibody drugs are poor. Due to their large size, their ability to penetrate and accumulate tissues is limited, and they are susceptible to enzymatic degradation mechanisms in the body, further reducing their pharmacokinetic properties. Summary of the invention

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

[0008] The invention 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) 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 human immunoglobulin E target: customizing a human immunoglobulin E protein target with a purity of at least 95%, and gradiently diluting 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: 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 human immunoglobulin E target is injected in sequence according to a concentration gradient. The interaction with the human immunoglobulin E 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 human immunoglobulin E target is identified, wherein lipoic acid is screened as a candidate drug with potential antagonistic effect;

[0014] (e) Drug molecular level verification: A human immunoglobulin E detection kit was used, with known human immunoglobulin E monoclonal antibody drugs as positive controls, to evaluate the antagonistic effect of lipoic acid on human immunoglobulin E, and the antagonistic activity of lipoic acid was verified by calculating the antagonism rate.

[0015] In a further preferred embodiment, the lipoic acid 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 in step (e) through ELISA detection.

[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] In a further preferred embodiment, the purity of the human immunoglobulin E target can be determined by SDS-PAGE or 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 molecule level verification step, the antagonism rate is calculated as follows: antagonism rate = 100% - (detection concentration / initial concentration) x 100%.

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

[0021] The present invention uses IgE as a target, performs high-throughput screening of drug molecules that can bind to IgE in the FDA drug library, verifies their antagonistic efficiency at the in vitro molecular level, and screens out a molecule that can antagonize IgE. The screened lipoic acid antagonistic efficiency has a significant antagonistic efficiency compared to common antagonists.

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

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

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

[0025] 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.

[0026] Figure 1 The lipoic acid signal curves collected when the IgE protein concentrations were 500nM, 250nM, 125nM, 62.5nM and 31.25nM, respectively.

[0027] Figure 2 This is the molecular structure of lipoic acid.

[0028] Figure 3 This is a dose-effect analysis diagram for omalizumab.

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

[0030] 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.

[0031] 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.

[0032] The present invention provides a method for screening an immunoglobulin E antagonist, comprising the following steps:

[0033] First, with IgE as the target, drug molecules that can bind to IgE are searched in the high-throughput screening FDA drug library; then, these drug molecules are tested for antagonistic efficiency in vivo to screen out molecules that can effectively antagonize IgE. In the specific process, 3067 customized FDA-approved compound drugs and IgE protein targets with measured purity are used for screening through microfluidic chips and PlexArray HT SPRi microarray analyzers, and finally small molecule compounds that meet the requirements, namely lipoic acid, are screened out through data analysis software. Then, the antagonistic effect of lipoic acid is detected and evaluated using the immunoglobulin E ELISA detection kit. The IgE monoclonal antibody drug adalimumab is also used as a positive control during the evaluation process. The entire screening process includes steps such as incubation of IgE and lipoic acid, immune response and color development, absorbance determination and concentration calculation.

[0034] More specifically, the following steps are included

[0035] (a) FDA drug library establishment: 3067 FDA-approved compound drugs were customized and stored in 1 mg / ml DMSO.

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

[0037] (c) Molecular screening with IgE 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 ranked by molecular affinity.

[0038] (d) Drug molecular level verification. The antagonistic effect of lipoic acid on IgE was evaluated using the human immunoglobulin E (IgE) ELISA test kit (Shenzhen Zike), with the IgE monoclonal antibody drug omalizumab as a positive control. The absorbance was detected using an enzyme marker, and the antagonism rate was calculated based on the detected concentration, antagonism rate = 100% - (detection concentration / initial concentration).

[0039] The amino acid sequence of IgE 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 steps for high-throughput screening of the FDA drug library chip using IgE as a target are as follows: (1) The prepared FDA drug library chip is taken out and placed in a 1% BSA solution for blocking for 2 h. The chip is washed with pure water and dried before being assembled into a microfluidic chip.

[0046] (2) Dilute the 1 mg / ml human immunoglobulin E (IgE) protein in PBST buffer in multiple dilutions.

[0047] (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.

[0048] (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.

[0049] (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.

[0050] (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.

[0051] Results reference Figure 1 and Figure 2 , and screened out a small molecule compound numbered S3996, lipoic acid. The results show that as the protein concentration increases, the detected binding signal becomes stronger, and its affinity ranks third among all small molecules. It is used as a candidate molecule for subsequent research.

[0052] In a preferred embodiment, the steps for analyzing the efficacy of the positive control omalizumab against IgE are as follows: (1) Add 50 μl of 10 μg / ml IgE protein to an EP tube, add different concentrations of omalizumab respectively, and incubate at 37°C for 1 hour.

[0053] (2) Add 100 μl of protein small molecule mixture or standard and 100 μl of HRP-labeled IgE antibody to the coated microwells pre-coated with IgE antibody, in turn, incubate at 37°C for 1 h, discard the solution, wash 5 times, and develop color with TMB.

[0054] (3) The absorbance at 450 nm was measured by an enzyme-labeled instrument, and the detection concentration was calculated after the standard curve was established. The antagonism rate was calculated based on the detection concentration, antagonism rate = 100% - (detection concentration / initial concentration).

[0055] Please refer to Figure 3The results are analyzed as shown in Table 1: The results of the dose-effect analysis of omalizumab showed that the antagonistic effect reached 22.96% when the concentration was 120 μg / ml.

[0056] Table 1:

[0057]

[0058] In a preferred embodiment, the steps of analyzing the efficacy of lipoic acid against IgE are as follows:

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

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

[0061] (3) The absorbance at 450 nm was measured by an enzyme-labeled instrument, and the detection concentration was calculated after the standard curve was established. The antagonism rate was calculated based on the detection concentration, antagonism rate = 100% - (detection concentration / initial concentration).

[0062] Please refer to Figure 4 As shown in Table 2, the results of the dose-effect analysis of S3996 lipoic acid showed that the concentration of the antagonist with the same antagonistic effect was 7.28 μg / ml.

[0063] Table 2:

[0064]

[0065] The above materials and reagents include:

[0066] FDA drug library chip was purchased from Selleck Chemicals, human immunoglobulin E was purchased from Biopsy Biotech, PBST buffer was purchased from Thermo Fisher Scientific, Gly-HCl buffer was purchased from Thermo Fisher Scientific, omalizumab was purchased from MedChemexpress Biotech, and lipoic acid was purchased from MedChemexpress Biotech, USA.

[0067] In summary, the present invention uses IgE as a target, high-throughput screening of drug molecules that can bind to IgE in the FDA drug library, and verifies its antagonistic efficiency at the in vitro molecular level, screens out a molecule that can antagonize IgE, and the thioctic acid antagonistic efficiency screened out has a significant antagonistic efficiency compared to common antagonists. First, the drug thioctic acid screened out by the present invention effectively avoids side effects such as acute allergic reactions and ensures the safety of patients. Secondly, by optimizing the drug structure, this technology reduces the immunogenicity of the CDR region, significantly reduces the production of anti-idiotypic antibodies, thereby extending the shelf life of the drug. Furthermore, thioctic acid has a stable structure and is not easily affected by environmental factors. Finally, thioctic acid is excellent in pharmacokinetics, has a smaller volume and stronger tissue penetration ability, and can resist enzyme degradation in the organism at the same time, thereby improving the bioavailability and efficacy of the drug. These advantages make this technology have broad application prospects in the treatment of IgE-related diseases.

[0068] Lipoic acid (LA), 5-[3-(1,2-dithioheterocyclic)]pentanoic acid, was first isolated from beef liver by Reed et al. in 1951. It is a trace element with multiple biological and pharmacological properties. It is a limiting essential nutrient required for cells to generate energy using energy substances such as carbohydrates. It is a pure natural antioxidant with the strongest antioxidant effect in nature. Because lipoic acid has an alkyl chain and a carboxyl end, it is both fat-soluble and water-soluble. It can pass through the blood-brain barrier and enter any tissue with 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, it removes oxygen free radicals and reactive oxygen and reduces the attack of free radicals on the body. In addition to a small amount of lipoic acid that can be synthesized by fatty acids and cysteine ​​in the body, the main lipoic acid in the human body needs to be ingested from food. The lipoic acid that enters the body can be quickly absorbed into the blood and accumulated in various tissues in the body, such as the heart, liver, and skeletal muscle. Studies have found that lipoic acid has been used to treat various chronic diseases, such as diabetes, liver fibrosis and neurodegenerative diseases. In vitro supplementation of lipoic acid can inhibit lipid oxidation of nerve tissue, prevent protein glycosylation, inhibit aldose reductase, and prevent the conversion of glucose or galactose into sorbitol. It is often used clinically to treat and relieve peripheral neuropathy caused by late diabetes; it can also strengthen the function of liver activity, improve energy metabolism rate, cooperate with other antioxidants to reduce free radical damage to liver cells, reduce inflammatory response, maintain liver function, and have a clearing effect on fat accumulation caused by non-alcoholic fatty liver disease, reducing the burden on the liver. In addition, lipoic acid is also a super antioxidant that can preserve and regenerate other antioxidants, balance blood sugar 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 a significant anti-inflammatory effect 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 endothelial human monocytes, 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] On the other hand, the present invention relates to a new use of lipoic acid, specifically using it as an antagonist of human immunoglobulin E (IgE). Lipoic acid can effectively bind to IgE through its unique chemical structure and biological activity, thereby inhibiting IgE-mediated immune responses and reducing the occurrence of allergic reactions.

[0071] On the other hand, the present invention provides an antiallergic pharmaceutical composition containing lipoic acid, which also includes a pharmaceutically acceptable carrier or excipient. The composition can effectively alleviate or prevent allergic reactions, including but not limited to hay fever, asthma and food allergies, through the antagonism of lipoic acid to IgE.

[0072] In another aspect, the present invention provides a method for preparing an antiallergic pharmaceutical composition containing lipoic acid, the method comprising mixing lipoic acid with a pharmaceutically acceptable carrier or excipient.

[0073] On the other hand, the present invention provides a method for treating or preventing allergic diseases using lipoic acid, the method comprising administering an effective amount of lipoic acid or a pharmaceutical composition thereof to a patient. The method can effectively alleviate or prevent the symptoms of allergic diseases and improve the quality of life of patients by inhibiting IgE-mediated immune responses.

[0074] On the other hand, the present invention also relates to the use of lipoic acid in the preparation of a drug for regulating the human immune system. Lipoic acid can regulate the function of the immune system and reduce the production and activity of IgE through its antioxidant, anti-inflammatory and immunomodulatory properties, thereby helping to maintain the balance and stability of the immune system.

[0075] On the other hand, the present invention provides an immunomodulator containing lipoic acid, which further comprises other immunomodulatory components and / or pharmaceutically acceptable carriers or excipients. The immunomodulator can effectively regulate the function of the immune system and reduce IgE-mediated immune responses through the synergistic effect of lipoic acid and other components, thereby preventing or alleviating the occurrence of allergic diseases.

[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 this field can change, modify, replace and modify the above embodiments within the scope of the present application.

Claims

1. A human immunoglobulin E antagonist, characterized in that The antagonist is lipoic acid.

2. The human immunoglobulin E antagonist according to claim 1, characterized in that The lipoic acid 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 human immunoglobulin E target: customizing a human immunoglobulin E protein target with a purity of at least 95%, and gradiently diluting 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; (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 human immunoglobulin E target is injected in sequence according to a concentration gradient. The interaction with the human immunoglobulin E 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 human immunoglobulin E target is identified, wherein lipoic acid is screened as a candidate drug with potential antagonistic effect; (e) Drug molecular level verification: A human immunoglobulin E detection kit was used, with known human immunoglobulin E monoclonal antibody drugs as positive controls, to evaluate the antagonistic effect of lipoic acid on human immunoglobulin E, and the antagonistic activity of lipoic acid was verified by calculating the antagonism rate.

3. The human immunoglobulin E antagonist according to claim 2, characterized in that: The lipoic acid 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 human immunoglobulin E 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.

5. The human immunoglobulin E antagonist according to claim 4, characterized in that: The purity of the human immunoglobulin E target can be determined by SDS-PAGE or other methods to ensure that it meets the screening requirements.

6. The human immunoglobulin E antagonist according to claim 5, 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%.

7. The human immunoglobulin E antagonist according to claim 6, characterized in that: Human immunoglobulin E is shown as SEQ ID NO:

1.

8. Use of lipoic acid as a human immunoglobulin E antagonist.

9. An antiallergic pharmaceutical composition containing lipoic acid.

10. A method for treating or preventing allergic diseases using lipoic acid.

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