Tumor necrosis factor alpha antagonist screening method and antagonist

Abstract

The invention relates to the technical field of medicinal chemistry, in particular to a screening method of a tumor necrosis factor alpha antagonist and the antagonist. The screening method comprises the following steps: by taking TNF-alpha as a target spot, screening drug molecules capable of being combined with TNF-alpha in an FDA drug library at high throughput; an antagonism efficiency test is carried out on the screened drug molecules in vivo, and molecules capable of antagonizing TNF-alpha are screened according to the expected antagonism efficiency. Drug molecules capable of being combined with TNF-alpha in an FDA drug library are screened in a high-throughput mode with the TNF-alpha 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 TNF-alpha is screened out, and compared with a common antagonist, the antagonism efficiency of the screened-out tretinoin has the remarkable antagonism efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a method for screening a tumor necrosis factor alpha antagonist and the antagonist. Background Art

[0002] Tumor necrosis factor α (TNF-α) is a multifunctional cytokine mainly secreted by activated immune cells such as macrophages, NK cells and T lymphocytes. After binding to TNF receptors (TNFR1 and TNFR2) on the cell surface, it can mediate a variety of biological effects, including inflammation, apoptosis, immune response, etc. TNF-α activates a series of complex intracellular signaling pathways by binding to its two receptors TNFR1 (also known as CD120a, p55) and TNFR2 (also known as CD120b, p75). TNFR1 is expressed on almost all types of cells and mainly mediates inflammatory responses and cell apoptosis-related signals by activating signaling pathways such as NF-κB and MAPKs. TNFR2 is mainly expressed on immune cells and mainly regulates cell survival, proliferation and immune response through signaling pathways such as PI3K / Akt.

[0003] TNF-α has a variety of biological functions and a relatively complex mechanism of action. Under normal physiological conditions, TNF-α is an important component of the immune system, which can help the body identify and eliminate infected or abnormal cells. It participates in maintaining immune homeostasis by binding to receptors on target cells, and enhances resistance to infection by activating neutrophils and platelets, enhancing the killing ability of macrophages / NK cells, and stimulating the immune system. However, in a disease state, abnormal expression or overactivation of TNF-α may cause a series of diseases. In autoimmune diseases such as rheumatoid arthritis and ankylosing spondylitis, TNF-α promotes the activation and infiltration of immune cells, activates inflammatory cells, promotes the release of cytokines, and induces tissue damage, thereby exacerbating the inflammatory response. In infectious diseases such as bacterial and viral infections, TNF-α can enhance the body's immune response and promote the elimination of pathogens. Therefore, blocking TNF-α has potential clinical application value.

[0004] Given the key role of TNF-α in a variety of diseases, biological agents composed mainly of TNF-α targeted inhibitors are an important component of TNF-α antagonists. Antibody drugs inhibit the role of TNF signaling pathways in diseases by blocking the interaction between TNF-α and TNFR1 / 2. Currently approved TNF-α inhibitors for clinical use include infliximab, enacept, adalimumab, certolizumab pegol and golimumab.

[0005] Biologic 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-like 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. 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 failure.

[0006] Retinamide, also known as fenretinide or 4-HRP, N-(4-hydroxyphenyl) retinamide, is a synthetic retinoid derivative, a new, effective and orally bioactive synthetic retinol (vitamin A) phenyl retinoic acid amide analogue, with potential anti-tumor and chemopreventive activity, and is a synthetic retinoid with anti-cancer activity. 4-HRP has low toxicity, which is due to the fact that 4-HRP is preferentially stored in adipose tissue, which can effectively prevent the accumulation of liver toxicity caused by 4-HRP treatment, and has great therapeutic advantages compared with the use of naturally derived vitamin A in clinical use.

[0007] Clinical studies have shown that 4-HRP is cytotoxic to a variety of human cancer cell lines in vitro. In addition, 4-HRP has anti-inflammatory, antiviral and immunomodulatory properties. There is evidence that 4-HRP inhibits the production of macrophage inflammatory mediators through the PPAR-γ pathway and reduces the inflammatory response of allergic asthma. In addition, 4-HRP can regulate the production of macrophage inflammatory mediators. In alcoholic liver disease, 4-HRP can reduce inflammation mediated by the TLR4-TNF-α axis in the intestine, thereby preserving the intestinal barrier and alleviating alcoholic liver disease. In a spontaneously hypertensive rat model, 4-HRP inhibits macrophage-mediated inflammation and lowers blood pressure by activating PPAR-γ.

[0008] TNF-α plays a key role in many diseases, and antibody drug therapy targeting TNF-α also has certain technical shortcomings.

[0009] (1) Antibody drugs have certain side effects.

[0010] 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.

[0011] (2) Antibody drugs have certain treatment risks.

[0012] 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.

[0013] (3) Antibody drugs are unstable and their structures are prone to change.

[0014] 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.

[0015] (4) Antibody drugs have poor pharmacokinetic properties.

[0016] 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. Summary of the invention

[0017] In view of the deficiencies of the prior art, the present invention provides a method for screening a tumor necrosis factor α antagonist and an antagonist.

[0018] The method for screening tumor necrosis factor α antagonists comprises the following steps:

[0019] S1: Targeting TNF-α, high-throughput screening of drug molecules that can bind to TNF-α in the FDA drug library;

[0020] S2: Conduct an in vivo antagonistic efficiency test on the drug molecules screened above, and screen out molecules that can antagonize TNF-α according to the expected antagonistic efficiency.

[0021] Preferably, in step S1:

[0022] 3067 FDA-approved compound drugs were customized and stored in 1 mg / ml DMSO; TNF-α protein target was customized, the purity of which was determined by SDS-PAGE, and TNF-α was diluted with deionized water according to the required gradient concentration; wherein the amino acid sequence of TNF-α is shown in SEQ ID NO: 1.

[0023] Preferably, in step S2:

[0024] 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 used for detection to verify the chip quality. The proteins to be tested were injected in sequence according to the concentration gradient. Finally, the collected signal data were analyzed using the data analysis software Data Processor Stand Alone. The results were sorted by molecular affinity to screen out small molecule compounds that met the requirements.

[0025] Preferably, the drug molecule screened out by the above method is retinamide.

[0026] Preferably, a human tumor necrosis factor-α (TNF-α) ELISA detection kit is used to detect the antagonistic effect of retinamide on TNF-α, and the antagonistic effect is evaluated.

[0027] Preferably, when evaluating the above antagonistic effect, the TNF-α monoclonal antibody drug adalimumab is also used as a positive control.

[0028] Another aspect of the present invention provides a tumor necrosis factor alpha antagonist, wherein the antagonist is retinamide. 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 retinoic acid phenolamide signal curves collected when the TNF-α protein concentrations were 500.0nM, 250.0nM, 125.0nM, 62.5nM and 31.2nM, respectively.

[0031] Figure 2 This is the molecular structure diagram of retinoic acid phenolamine.

[0032] Figure 3This is a dose-effect analysis diagram of the positive control adalimumab with concentrations of 0, 2000, 8000, 32000, 128000, 512000, and 2048000 μg / L.

[0033] Figure 4 This is a dose-effect analysis diagram for retinoic acid phenolamide concentrations of 0, 8.95, 35.80, 143.20, 572.78, 2291.13, and 9164.51 μg / L. DETAILED DESCRIPTION

[0034] 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.

[0035] 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.

[0036] The present invention provides a method for screening a tumor necrosis factor alpha antagonist, comprising the following steps:

[0037] (1) Establishment of FDA drug library: 3067 FDA-approved compound drugs were customized and stored in 1 mg / ml DMSO.

[0038] (2) Preparation of TNF-α target. A TNF-α protein target was customized, and its purity was determined to be 95% by SDS-PAGE. TNF-α was gradiently diluted with deionized water to 500 nM, 250 nM, 125 nM, 62.5 nM and 31.25 nM. The amino acid sequence of TNF-α is shown in SEQ ID NO: 1.

[0039] The amino acid sequence of TNF-α is:

[0040] SEQ ID NO: 1: VRSSSRTPSDKPVAHVVANP QAEGQLQWLN RRANALLANG VELRDNQLVVPSEGLYLIYS

[0041] 61QVLFKGQGCP STHVLLTHTI SRIAVSYQTK VNLLSAIKSP CQRETPEGAE AKPWYEPIYL

[0042] 121GGVFQLEKGD RLSAEINRPD YLDFAESGQV YFGIIAL

[0043] (3) Molecular screening with TNF-α 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.

[0044] (4) Drug molecular level verification. The antagonistic effect of retinolamide on TNF-α was evaluated using the human tumor necrosis factor-α (TNF-α) ELISA test kit (Shenzhen Zike), with adalimumab, a TNF-α monoclonal antibody, as the positive control. The absorbance was detected using an ELISA instrument, and the antagonism rate was calculated based on the detection concentration, antagonism rate = 100% - (detection concentration / initial concentration).

[0045] In a preferred embodiment, a high-throughput screening of the FDA drug library chip is performed targeting TNF-α. The screening steps 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 tumor necrosis factor-α (TNF-α) protein concentration in multiples using PBST buffer.

[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] Please refer to Figure 1 and Figure 2 , and analyzed the results: a small molecule compound numbered S5233, fenretinide, was screened out. The results show that as the protein concentration increases, the detected binding signal becomes stronger, and its affinity ranks second among all small molecules. It is used as a candidate molecule for subsequent antagonism rate and other studies.

[0053] In a preferred embodiment, the analysis of the efficacy of the positive control adalimumab against TNF-α is performed in the following steps:

[0054] (1) Add 50 μl of 100 pg / ml TNF-α protein to an EP tube, add different concentrations of adalimumab, 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 TNF-α antibody to the coated microwells pre-coated with TNF-α antibody, in turn, incubate at 37°C for 1 h, discard the solution, wash 5 times, and develop color with TMB.

[0056] (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 according to the detection concentration, antagonism rate = 100% - (detection concentration / initial concentration), as shown in Table 1 below.

[0057] Table 1:

[0058]

[0059] Please refer to Figure 3 , and analyzed the results: The results of the dose-effect analysis of adalimumab subcutaneously injected with 40 mg found that the antagonistic effect reached 16.05% when the monoclonal antibody concentration was 8.00 μg / ml (the used concentration).

[0060] In a preferred embodiment, the analysis of the efficacy of retinol amine in antagonizing TNF-α is performed in the following steps:

[0061] (1) Add 50 μl of 100 pg / ml TNF-α protein to an EP tube and add different concentrations of retinol amide, and incubate at 37°C for 1 h.

[0062] (2) Add 100 μl of protein small molecule mixture or standard and 100 μl of HRP-labeled TNF-α antibody to the coated microwells pre-coated with TNF-α antibody, in turn, incubate at 37°C for 1 h, discard the solution, wash 5 times, and develop color with TMB.

[0063] (3) Use an enzyme-labeled instrument to measure the absorbance at 450 nm, and calculate the detection concentration after establishing a standard curve. And calculate the antagonism rate based on the detection concentration, antagonism rate = 100% - (detection concentration / initial concentration). Please refer to Table 2.

[0064] Table 2:

[0065]

[0066] Please refer to Figure 4 , and analyzed the results: The results of the dose-effect analysis of S5233 retinoic acid phenolamine found that the concentration of the antagonist with the same antagonistic effect was 48.11μg / L.

[0067] The above materials and reagents include:

[0068] FDA drug library chip was purchased from Selleck Chemicals, tumor necrosis factor-α was purchased from Beijing Sino Biological Technology Co., Ltd., PBST buffer was purchased from Thermo Fisher Scientific, Gly-HCl buffer was purchased from Thermo Fisher Scientific, human tumor necrosis factor-α (TNF-α) ELISA detection kit was purchased from Shenzhen Zike Biotechnology Co., Ltd., adalimumab was purchased from MedChemexpress Biotechnology Co., Ltd., USA, and retinamide was purchased from MedChemexpress Biotechnology Co., Ltd., USA.

[0069] In summary, the present invention uses TNF-α as a target, conducts high-throughput screening of drug molecules that can bind to TNF-α in the FDA drug library, and verifies their antagonistic efficiency at the in vitro molecular level, thereby screening out a molecule that can antagonize TNF-α, and the screened retinoic acid phenolamine antagonistic efficiency has a significant antagonistic efficiency compared with ordinary antagonists.

[0070] 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 method for screening a tumor necrosis factor α antagonist, characterized in that: The method for screening tumor necrosis factor α antagonists comprises the following steps: S1: Targeting TNF-α, high-throughput screening of drug molecules that can bind to TNF-α in the FDA drug library; S2: Conduct an in vivo antagonistic efficiency test on the drug molecules screened above, and screen out molecules that can antagonize TNF-α according to the expected antagonistic efficiency.

2. The screening method according to claim 1, characterized in that In step S1: 3067 FDA-approved compound drugs were customized and stored in 1 mg / ml DMSO; TNF-α protein target was customized, the purity of which was determined by SDS-PAGE, and TNF-α was diluted with deionized water according to the required gradient concentration; wherein the amino acid sequence of TNF-α is shown in SEQ ID NO:

1.

3. The screening method according to claim 2, characterized in that In step S2: 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 used for detection to verify the chip quality. The proteins to be tested were injected in sequence according to the concentration gradient. Finally, the collected signal data were analyzed using the data analysis software Data Processor Stand Alone. The results were sorted by molecular affinity to screen out small molecule compounds that met the requirements.

4. The method for screening tumor necrosis factor α antagonists according to claim 3, characterized in that: The drug molecule screened out by the above method is retinoic acid phenolamine.

5. The method for screening tumor necrosis factor α antagonists according to claim 4, characterized in that: The human tumor necrosis factor-α (TNF-α) ELISA detection kit was used to detect the antagonistic effect of retinamide on TNF-α and evaluate the antagonistic effect.

6. The method for screening tumor necrosis factor α antagonists according to claim 5, characterized in that: When evaluating the above antagonistic effect, TNF-α monoclonal antibody adalimumab was also used as a positive control.

7. A tumor necrosis factor α antagonist, characterized in that: The antagonist is retinamide.

Images