A screening method for human immunoglobulin E antagonist and antagonist

Abstract

The present invention relates to a screening method for a human immunoglobulin E antagonist and an antagonist, comprising the following steps: S1, customizing a specific human immunoglobulin E as a target, and customizing an FDA drug library as a drug molecule library; S2, using the target to perform high-throughput molecular screening in the drug molecule library, and screening out specific drug molecules that can combine with the target from the drug molecule library; S3, detecting the antagonistic effect of the specific drug molecule on the target, and performing a dosage effect evaluation of the specific drug molecule antagonizing IgE, wherein the antagonist is pinaverium bromide.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a method for screening a human immunoglobulin E antagonist and the antagonist. Background Art

[0002] Human immunoglobulin E is abbreviated as IgE. Human immunoglobulin E refers to the production of antibodies that can specifically bind to the corresponding antigens when B lymphocytes are transformed into plasma cells under antigen stimulation. It is an immunoglobulin that plays an important role in the human immune system. 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 content of IgE is extremely low, accounting for about 0.002% of the total immunoglobulin in serum. The important role of IgE in immune response and inflammatory response. Under normal physiological function, IgE is highly specific and mainly acts as a key molecule that mediates allergic reactions. It can recognize and bind to allergens that the body contacts, such as pollen, dust mites, animal dander, food, drugs and other allergens. IgE will quickly recognize and bind to these allergens, thereby activating immune cells such as mast cells and eosinophils, triggering a series of immune responses, including inflammatory mediators such as histamine release, and ultimately leading to allergic reactions such as skin redness, sneezing or asthma. Under normal conditions, IgE levels remain within a relatively stable range to ensure that they respond to potentially harmful substances without triggering excessive or unnecessary immune responses.

[0003] In allergic diseases such as allergic rhinitis and allergic asthma, IgE mainly binds to high-affinity receptors on the surface of mast cells and basophils. The IgE-FcεR1 complex binds tightly, and the bound IgE-FcεR1 complex causes changes in the receptor conformation, which in turn activates protein kinases in the cell, such as Src family kinases and Syk kinases, and further triggers changes in downstream signaling pathways, such as the MAPK signaling pathway and the NF-κB signaling pathway. The combination of is the initiating link of allergic reaction. Blocking this link is crucial to the treatment of allergic diseases.

[0004] Due to the key role of IgE in many diseases, biological agents mainly composed of IgE targeted inhibitors are an important component of IgE antagonists. Antibody drugs such as IgE antagonists block IgE-mediated allergic reactions by blocking the binding of IgE to high-affinity receptors (FcεRI) on the surface of effector cells (such as mast cells and eosinophils). This mechanism of action can effectively reduce the activation of mast cells and eosinophils, thereby reducing the release of inflammatory mediators, thereby alleviating allergic symptoms. Currently approved IgE inhibitors for clinical use include omalizumab, legrezumab, etc. However, biological antibody drugs have certain side effects or treatment risks. Acute reactions after injection of monoclonal antibodies can be caused by a variety of mechanisms, including acute allergic (IgE-mediated) and anaphylactic reactions to monoclonal antibodies, serum sickness, tumor lysis syndrome, and cytokine release syndrome. Clinical manifestations include local skin reactions at the injection site, fever, and influenza-like syndrome. Moreover, the humanization of antibody drugs does not completely eliminate immunogenicity. Even the CDR region of the humanized antibody still has strong immunogenicity, producing anti-idiotypic antibodies and causing drug ineffectiveness.

[0005] IgE plays a key role in many diseases, and antibody drug therapy targeting IgE also has certain technical disadvantages:

[0006] (1) Antibody drugs have certain side effects

[0007] Acute reactions after injection of antibody drugs can be caused by a variety of mechanisms, including acute allergic (IgE-mediated) and anaphylactoid reactions to monoclonal antibodies, serum sickness, tumor lysis syndrome, and cytokine release syndrome. Clinical manifestations include local skin reactions at the injection site, fever, and flu-like syndrome.

[0008] (2) Antibody drugs have certain treatment risks

[0009] The humanization of antibody drugs does not completely eliminate immunogenicity. Even the CDR region of the humanized antibody still has strong immunogenicity, producing anti-idiotypic antibodies and causing drug ineffectiveness.

[0010] (3) Antibody drugs are unstable and their structures are prone to change

[0011] Biomacromolecules such as monoclonal antibodies have unique three-dimensional structures. The complex balance of interactions between amino acid functional groups and the external environment, within molecules and between molecules determines the folded structure. Since the folded structure is in a dynamic equilibrium state, any factor that changes the balance of interactions may cause structural changes, resulting in an unstable state of the macromolecule. Since the tertiary structure of biological products is susceptible to environmental physical stress, structural changes in monoclonal antibodies may occur at any stage of the production process, from initial protein expression to processing to storage.

[0012] (4) Poor pharmacokinetic properties of antibody drugs

[0013] Most monoclonal antibodies are large in size and have limited ability to penetrate and accumulate in tissues, and are likely to be confined to the interstitial space after injection. Biotherapeutics can reach the blood circulation in two ways: through capillaries or lymphatic vessels. However, absorption through capillaries has been reported to rely on passive transport and is limited to compounds with a molecular weight below 16 kDa. Therefore, most biotherapeutic drugs cannot be transported through the capillary route, but rely on the lymphatic system. The pharmacokinetic properties of most types of protein drugs are further reduced due to enzymatic degradation mechanisms in the body, and proteins and monoclonal antibodies are prone to enzymatic degradation.

[0014] Therefore, it is particularly important to find drug molecules that can antagonize IgE. Summary of the invention

[0015] Based on this, it is necessary to provide a screening method for human immunoglobulin E antagonists and antagonists.

[0016] In one aspect, an embodiment of the present invention provides a method for screening a human immunoglobulin E antagonist. The method for screening a human immunoglobulin E antagonist comprises the following steps:

[0017] S1. Customize specific human immunoglobulin E as a target and customize the FDA drug library as a drug molecule library for backup;

[0018] S2. Use the target to perform high-throughput molecular screening in the drug molecule library to screen out specific drug molecules that can bind to the target from the drug molecule library;

[0019] S3. Detect the antagonistic effect of the specific drug molecule on the target, and evaluate the efficacy of the specific drug molecule in antagonizing IgE.

[0020] Preferably, in step S1, 3067 FDA-approved compound drugs are customized and dissolved and stored in 1 mg / ml DMSO; an IgE target is customized, and its purity is 95% as determined by SDS-PAGE; the IgE target is diluted with deionized water in a gradient concentration; wherein the amino acid sequence of the IgE target is as shown in SEQ ID NO: 1.

[0021] Preferably, in step S2, the FDA drug library chip is assembled into a microfluidic chip and installed in the PlexArray HTSPRi microarray analyzer; then, the IgE target to be tested is sampled sequentially according to the concentration gradient, and finally, the collected signal data is analyzed using the data analysis software Data Processor Stand Alone, the results are sorted by molecular affinity, and molecular compounds that meet the requirements are screened out from the drug molecules that can bind to the target.

[0022] Preferably, the drug molecule screened out by the screening method is pinaverium bromide.

[0023] Preferably, before the IgE target to be tested is injected sequentially according to the concentration gradient, a positive sample is injected and tested in advance, and the chip quality is verified based on whether a positive response signal of the positive sample is detected.

[0024] Preferably, the positive sample is rapamycin.

[0025] Preferably, in step S3, the antagonistic effect of pinaverium bromide on IgE is detected using an IgE ELISA detection kit, and the antagonistic effect is evaluated using the IgE monoclonal antibody drug omalizumab as a positive control.

[0026] Preferably, the antagonism rate is the quotient of the detection concentration and the initial concentration.

[0027] On the other hand, a human immunoglobulin E antagonist is provided, wherein the antagonist is pinaverium bromide.

[0028] 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 small molecule drug, pinaverium bromide, that can antagonize IgE. The screened small molecule drug, pinaverium bromide, provides a new idea for studying the function of IgE. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The above and other purposes, features and advantages of the present invention will become more clear 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, and the focus is on illustrating the main purpose of the present invention.

[0030] Figure 1 The embodiment of the present invention is a curve diagram of the pinaverium bromide signal collected by sampling when the gradient concentrations of IgE protein are 500.0 nM, 250.0 nM, 125.0 nM, 62.5 nM and 31.2 nM respectively;

[0031] Figure 2 It is the dose-effect analysis diagram of the positive control omalizumab at concentrations of 0, 18.75, 37.5, 75, 150, 300, and 600 μg / ml;

[0032] Figure 3 It is a dose-effect analysis diagram of vepivamide bromide concentrations of 0, 157.19, 628.75, 2515, 10060, 40240, and 160960 μg / L;

[0033] Figure 4 This is the molecular structure of pinaverium bromide. DETAILED DESCRIPTION

[0034] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0035] like Figure 1-3 As shown, a method for screening a human immunoglobulin E antagonist and the antagonist comprises the following steps:

[0036] Step 1: Establish a drug molecule library consisting of 3067 FDA-approved compound drugs, dissolve and store them in 1 mg / ml DMSO. At the same time, customize human immunoglobulin E as an IgE target, and its purity is 95% as determined by SDS-PAGE. Use deionized water to dilute IgE to 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM. The amino acid sequence of the IgE target is shown in SEQ ID NO: 1.

[0037] The amino acid sequence of IgE is:

[0038] SEQ ID NO: 1:

[0039] 1 CADSNPRGVSAYLSRPSPFDLFIRKSPTITCLVVDLAPSKGTVNLTWSRASGKPVNHSTR

[0040] 61 KEEKQRNGTLTVTSTLPVGTRDWIEGETYQCRVTHPHLPRALMRSTTKTSGPRAAPEVYA

[0041] 121 FATPEWPGSRDKRTLACLIQNFMPEDISVQWLHNEVQLPDARHSTTQPRKTKGSGFFVFS

[0042] 181 RLEVTRAEWEQKDEFICRAVHEAASPSQTVQRAVSVNPGK

[0043] Step 2: Use the target to perform high-throughput molecular screening in the drug molecule library to screen out specific drug molecules that can bind to the target from the drug molecule library:

[0044] High-throughput molecular screening with IgE as the target: The FDA drug library chip is assembled into a microfluidic chip and installed in the PlexArray HT SPRi microarray analyzer, and the positive sample rapamycin is used for detection to verify the chip quality. The proteins to be tested are injected in sequence according to the above concentration gradient, and finally the collected signal data is analyzed using the data analysis software Data Processor Stand Alone, and the results are sorted by molecular affinity.

[0045] Step 3: Detect the antagonistic effect of the specific drug molecule on the target, and evaluate the efficacy of the specific drug molecule in antagonizing IgE. The screened drug molecule is pinaverium bromide.

[0046] The selected drug molecules were verified at the drug molecule level: the human immunoglobulin E (IgE) ELISA test kit (Shenzhen Zike) was used to test the antagonistic effect of pinaverium bromide on IgE, and the IgE monoclonal antibody drug omalizumab was used as a positive control. The absorbance was detected by an enzyme-labeled instrument, and the antagonism rate was calculated based on the detection concentration, antagonism rate = 100% - (detection concentration / initial concentration).

[0047] like Figure 4 As shown, pinaverium bromide has a new application as a human immunoglobulin E antagonist.

[0048] In a preferred embodiment, high-throughput screening of the FDA drug library chip is performed using IgE as a target, and the steps are as follows:

[0049] (1) The prepared FDA drug library chip was taken out and sealed in a 1% BSA solution for 2 h. The chip was washed with pure water and dried before being assembled into a microfluidic chip.

[0050] (2) Dilute 1 mg / ml human immunoglobulin E (IgE) protein in PBST buffer in multiple dilutions.

[0051] (3) The microfluidic chip was installed in the PlexArray HT SPRi microarray analyzer, and a 100 nM FKBP12 positive sample was first flowed in 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.

[0052] (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 samples 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.

[0053] (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.

[0054] (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 pinaverium bromide numbered S6473 was screened out.

[0055] As attached Figure 1 As shown, it can be seen that as the gradient of IgE protein concentration increases, the binding signal collected by the analysis software Data ProcessorStand Alone becomes stronger. Pinaverium bromide ranks second in affinity among all small molecules, and it is used as a candidate molecule for subsequent research. Figure 4 This is the molecular structure of pinaverium bromide.

[0056] In a preferred embodiment, the positive control omalizumab is subjected to a dose-effect analysis on antagonism of IgE, and the steps are as follows:

[0057] (1) Add 50 μl of 10 μg / ml IgE protein to the EP tube, add different concentrations of omalizumab, and incubate at 37°C for 1 h.

[0058] (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.

[0059] (3) Use an enzyme-labeled instrument to measure the absorbance at 450 nm and calculate the detection concentration after establishing a standard curve.

[0060] The antagonism rate was calculated according to the detected concentration, antagonism rate = 100% - (detection concentration / initial concentration), and the results are shown in Table 1 below:

[0061] Table 1 Antagonism rate of different concentrations of omalizumab against IgE

[0062]

[0063] refer to Figure 2 , and the results were analyzed. It was found that the dose-effect analysis of omalizumab found that the antagonistic effect reached 22.96% when the concentration of omalizumab was 120 μg / ml.

[0064] In a preferred embodiment, the dose-effect analysis is performed using pinaverium bromide to antagonize IgE, and the steps are as follows:

[0065] (1) Add 50 μl of 10 μg / ml IgE protein to the EP tube, add different concentrations of pinaverium bromide, and incubate at 37°C for 1 h.

[0066] (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.

[0067] (3) Use an enzyme-labeled instrument to measure the absorbance at 450 nm and calculate the detection concentration after establishing a standard curve.

[0068] The antagonism rate was calculated according to the detected concentration, antagonism rate = 100% - (detection concentration / initial concentration), and the results are shown in Table 2 below:

[0069] Table 2 Antagonism rate of different concentrations of pinaverium bromide against IgE

[0070]

[0071] refer to Figure 3 , and the results were analyzed. It was found that the dose-effect analysis results of S6473 pinaverium bromide found that the concentration of the antagonist with the same antagonistic effect was 2.42 μg / ml.

[0072] The above materials and reagents: FDA drug library chip was purchased from Selleck Chemicals. Human immunoglobulin E was purchased from Biopsys Biotech, PBST buffer was purchased from Thermo Fisher Scientific, and Gly-HCl buffer was purchased from Thermo Fisher Scientific. Human immunoglobulin E (IgE) ELISA test kit was purchased from Shenzhen Zike Biotechnology Co., Ltd., Omalizumab was purchased from MedChemexpress Biotech, USA. Pinaverium bromide was purchased from MedChemexpress Biotech, USA.

[0073] In summary, the present invention uses IgE as a target, conducts high-throughput screening of drug molecules that can bind to IgE in the FDA drug library, and verifies their antagonistic efficiency at the in vitro molecular level, thereby screening out a molecule that can antagonize IgE, and the screened pinaverium bromide antagonistic efficiency has a significant antagonistic efficiency compared to ordinary antagonists.

[0074] Pinaverium bromide is a calcium antagonist with highly selective antispasmodic effect on the gastrointestinal tract. It is used to treat irritable bowel syndrome, pain related to intestinal dysfunction, abnormal intestinal motility and discomfort. The mechanism of action of pinaverium bromide on smooth muscle is the same as other calcium antagonists, but it has a highly selective effect on colonic smooth muscle. Pinaverium bromide blocks the influx of calcium ions into the smooth muscle cells of the intestinal wall, preventing excessive muscle contraction and achieving antispasmodic effect. It can eliminate the high reactivity of intestinal smooth muscle and increase intestinal motility. Its chemical name is N (2-bromo-4,5-dimethylbenzyl)-N (2 [dimethyl-2-demethylpinyl] ethoxy) ethyl) morpholinium bromide. The mechanism of action of pinaverium bromide on smooth muscle is the same as other calcium antagonists, but it has a highly selective effect on colonic smooth muscle. Pinaverium bromide blocks the influx of calcium ions into the smooth muscle cells of the intestinal wall, preventing excessive muscle contraction and achieving antispasmodic effect. It can eliminate the high reactivity of intestinal smooth muscle and increase intestinal motility. Pinaverium bromide has very low affinity for cardiovascular smooth muscle cells. A single oral dose of 1200 mg per day will not cause changes in blood pressure. Pinaverium bromide does not affect the pressure of the lower esophageal cardia sphincter, nor does it cause duodenal reflux, but it has a relaxing effect on the sphincter of Oddi. In vitro studies have shown that this product has a dose-dependent inhibitory effect on smooth muscle contraction caused by barium chloride, acetylcholine, norepinephrine, carbachol and electrical stimulation.

[0075] The mode of action of pinaverium bromide in the body is achieved through the activation of calcium channels. There are two different types of calcium channels on the cell membrane of gastrointestinal smooth muscle: ① receptor management channels, which are activated by changes in the structure of specific membrane-bound receptors; ② voltage management channels, whose opening and closing depend on the membrane potential. In vitro study results show that pinaverium bromide produces antispasmodic effects by blocking voltage-dependent calcium currents. Pinaverium bromide is a highly polar quaternary ammonium compound with poor oral absorption. Only less than 10% of the drug enters the blood and is almost entirely bound to plasma proteins. After oral administration of 100 mg, tmax is 0.5 to 3 hours and t1 / 2 is about 1.5 hours. It is rapidly metabolized and mainly excreted from the feces through the liver and gallbladder. The LD50 of this product for intravenous injection in mice is 37 to 66 mg / kg, and the oral LD50 is 1400 mg / kg. It can be seen that pinaverium bromide has good drug safety performance, and the new application of pinaverium bromide as an IgE antagonist has good development prospects.

[0076] The above are only preferred embodiments of the present invention, and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Use of pinaverium bromide in the preparation of human immunoglobulin E antagonists.

2. A method for screening human immunoglobulin E antagonists, characterized in that: The method for screening human immunoglobulin E antagonists comprises the following steps: S1. Customize specific human immunoglobulin E as a target and customize the FDA drug library as a drug molecule library for backup; In step S1, 3067 FDA-approved compound drugs are customized and dissolved and stored at 1 mg / ml DMSO; an IgE target is customized, and its purity is 95% as determined by SDS-PAGE; the IgE target is diluted with deionized water in a gradient concentration; wherein the amino acid sequence of the IgE target is shown in SEQ ID NO: 1; S2, using the target to perform high-throughput molecular screening in the drug molecule library, and screening out specific drug molecules that can bind to the target from the drug molecule library; in step S2, the FDA drug library chip is assembled into a microfluidic chip and installed in the PlexArray HT SPRi microarray analyzer; then, the IgE target to be tested is injected in sequence according to the concentration gradient, and finally, the collected signal data is analyzed using the data analysis software Data Processor Stand Alone, and the results are sorted by molecular affinity, and molecular compounds that meet the requirements are screened out from the drug molecules that can bind to the target; Before the IgE target to be tested is injected sequentially according to the concentration gradient, a positive sample is injected and tested in advance, and the chip quality is verified according to whether a positive response signal of the positive sample is detected; S3, detecting the antagonistic effect of the specific drug molecule on the target, and evaluating the efficacy of the specific drug molecule in antagonizing IgE, wherein the drug molecule screened out by the screening method is pinaverium bromide; Pinaverium bromide as a human immunoglobulin E antagonist.

3. The screening method according to claim 2, characterized in that The positive sample was rapamycin.

4. The screening method according to claim 2, characterized in that In step S3, the antagonistic effect of pinaverium bromide on IgE is detected using an IgE ELISA detection kit, the antagonistic effect is evaluated using the IgE monoclonal antibody drug omalizumab as a positive control, the absorbance at 450 nm is detected using an enzyme reader, the detection concentration is calculated after establishing a standard curve, and the antagonism rate is calculated based on the detection concentration.

5. The screening method according to claim 4, characterized in that The antagonism rate is the quotient of the detection concentration and the initial concentration.

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