TIM-3 micromolecule binding agent, screening method and application

Abstract

The invention relates to the technical field of biological medicine, in particular to a TIM-3 small molecule binding agent, a screening method and application, the TIM-3 small molecule binding agent comprises a small molecule compound bound with TIM-3 protein, and compared with an antibody drug, the small molecule compound is small in molecular weight, high in penetrating power and capable of easily penetrating through a cell membrane, so that the TIM-3 protein binding agent can be used for binding TIM-3 protein. The secretion of cell factors such as gamma interferon, interleukin-2, tumor necrosis factor-alpha, granzyme B and the like by T cells is effectively enhanced, so that the compound is expected to be applied to the preparation of immunomodulatory preparations; according to the screening method, firstly, compounds in a compound library are preliminarily screened through a molecular docking platform, then the binding capacity of the screened compounds and TIM-3 protein is verified through the surface ion resonance technology, small molecule compounds with pharmaceutical potential are screened out, and the screening method greatly saves the time and cost of drug research and development.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a TIM-3 small molecule binder, a screening method and an application thereof. Background Art

[0002] Immune checkpoint blockade (ICB) therapy is a method for treating tumors, which has shown significant therapeutic effects in the treatment of various types of tumors. The basic principle of immune checkpoint blockade therapy is based on the activation mechanism of immune T cells. Immunoinhibitory receptors are expressed on the surface of T cells, and their ligands are expressed on the surface of tumor cells. The binding of immunoinhibitory receptors to their ligands can cause T cells to fail and fail to kill tumor cells normally, so that tumor cells can escape the host's immune surveillance. Immune checkpoint blockade therapy based on immunoinhibitory receptors and their ligands inhibits the binding of the two, thereby enhancing the host immune system's killing effect on tumor cells.

[0003] Currently, therapeutic antibodies targeting programmed death receptor-1 (PD-1) and cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) have achieved significant and lasting therapeutic effects in a variety of tumor types by restoring the effector function of exhausted T cells. However, a large number of cancer patients are ineffective against PD-1 or CTLA-1 blockade, or initially respond but eventually develop resistance. Therefore, the development of other immune checkpoint blockade drugs is urgently needed.

[0004] TIM-3 (T cell immunoglobulin and mucin domain-containing protein 3) is a type I transmembrane protein that is highly expressed in dysfunctional and exhausted T cells and has been widely considered to be a promising immune checkpoint and target for tumor immunotherapy. TIM-3 consists of a single N-terminal immunoglobulin variable (IgV) domain, a glycosylated mucin-like domain, a transmembrane region, and a C-terminal intracellular tail. Currently, four different ligands of TIM-3 have been reported, including galactose binding protein-9, phosphatidylserine (PtdSer), high mobility group box B1 (HMGB1), and carcinoembryonic antigen associated cell adhesion molecules 1 (CEACAM1). The interaction between these ligands and TIM-3 protein has important clinical significance for regulating the immune system to inhibit tumor growth.

[0005] In mouse models, antibodies against TIM-3 were able to reverse T cell exhaustion and inhibit tumor growth. In addition, CD8+ T cells expressing PD-1 and TIM-3 showed the most exhausted phenotype in melanoma patients and tumor mouse models, with significantly impaired proliferation and cytokine production. Upregulation of TIM-3 is associated with resistance to PD-1 inhibitors. Combination therapy of TIM-3 and PD-1 blockade is more effective than monotherapy in enhancing T cell function to inhibit tumor growth. Although antibodies are widely used to block immune checkpoint pathways, and several TIM-3 antibodies are currently in preclinical development, the development and application of antibody drugs are limited by the complex preparation process, high R&D costs, the need for low-temperature storage, and inconvenience in use. Summary of the invention

[0006] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the first object of the present invention is to provide a TIM-3 small molecule binder; the second object of the present invention is to provide a screening method for a TIM-3 small molecule binder; the third object of the present invention is to provide an application of a TIM-3 small molecule binder.

[0007] In order to achieve the first purpose, the technical solution adopted by the present invention is: A TIM-3 small molecule binder includes a small molecule compound that binds to a TIM-3 protein, and its structural formula is as follows: ; Wherein, R1 is H or a halogen atom, R2 and R3 are each independently H or OH, and R4 is an alkyl group; TIM-3 is a negatively regulated immune checkpoint protein and a member of the TIM family. It is expressed on the surface of T cells, T regulatory cells and innate immune cells, and exerts immunosuppressive effects mainly by inhibiting T helper cells, inducing CD8+T cell exhaustion, and promoting T regulatory cells to become immunosuppressive cell populations. TIM-3 is one of the most studied immunotherapy targets. For patients who have no response to or develop resistance to anti-PD-1 monoclonal antibodies, high expression of TIM-3 may lead to immune escape. The small molecule binder provided by the present invention can bind to the TIM-3 protein, prevent the immune escape caused by high expression of TIM-3, and help the body's immune system to exert its immune activation effect.

[0008] Furthermore, the small molecule compound is selected from any one or more of the following structural formulas: , and .

[0009] Furthermore, it also includes at least one of a salt, a hydrate and a precursor compound of the small molecule compound.

[0010] Furthermore, it also includes one or more of pharmaceutically acceptable adjuvants, excipients and carriers.

[0011] Furthermore, it includes single preparations or compound preparations with small molecule compounds as active ingredients.

[0012] In order to achieve the second purpose, the technical solution adopted by the present invention is: A method for screening a TIM-3 small molecule binder, for screening any of the TIM-3 small molecule binders described above, comprising the following steps: S1. Screen the compounds in the compound library through the molecular docking platform to obtain compounds with a docking score lower than -4; S2. Use surface ion resonance technology to detect the binding force between the compound obtained in step S1 and the TIM-3 protein, and select small molecule compounds with a binding force greater than or equal to 5.8 as TIM-3 small molecule binders.

[0013] In order to achieve the third purpose, the technical solution adopted by the present invention is: An application of a TIM-3 small molecule binder, such as any of the TIM-3 small molecule binders described above, for preparing an immunomodulatory preparation. An immunomodulatory preparation is a class of drugs or substances that prevent or treat immune-related diseases or inflammatory diseases by regulating the function of the body's immune system.

[0014] Furthermore, the immunomodulatory preparation includes at least one of an oral preparation, an injection preparation and a transdermal preparation.

[0015] Furthermore, the immunomodulatory preparation is used to enhance the function of the body's effector T cells in secreting cytokines.

[0016] Furthermore, the immunomodulatory preparation is used in tumor patients.

[0017] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The present invention provides a TIM-3 small molecule binder, including a small molecule compound that binds to the TIM-3 protein. The small molecule compound included in the binder has a small molecular weight, strong penetrating power, can easily penetrate the cell membrane, and can effectively enhance the function of T cells to secrete cytokines such as interferonγ (IFN-γ), interleukin 2 (IL-2), tumor necrosis factor α (TNF-α) and granzyme B (GZMB). Compared with antibody drugs, small molecule immune checkpoint inhibitors or binders have natural advantages, such as small molecular weight, strong penetration into tissues, convenient medication, convenient storage, low preparation cost, and potential for more excellent ability to inhibit tumor growth and migration, as well as good biosafety. Therefore, they are expected to be used in the preparation of immunomodulatory preparations; The present invention provides a method for screening TIM-3 small molecule binders. First, the compounds in the compound library are preliminarily screened through a molecular docking platform, and then the binding ability of the screened compounds to the TIM-3 protein is detected using surface ion resonance technology to screen out compounds with pharmaceutical potential. This screening method greatly saves time and cost for drug research and development.

[0018] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1It is a schematic diagram of the binding of the small molecule compound T16 provided in Example 1 of the present invention to the TIM-3 protein.

[0021] Figure 2 It is the binding force between the small molecule compound T16 of different concentrations provided in Example 2 of the present invention and the TIM-3 protein.

[0022] Figure 3 It is the expression level of IFN-γ after different concentrations of the small molecule compound T16 provided in Example 3 of the present invention acted on human T cells for 24 hours.

[0023] Figure 4 It is the expression level of TNF-α after different concentrations of the small molecule compound T16 provided in Example 3 of the present invention acted on human T cells for 24 hours.

[0024] Figure 5 The expression level of IL-2 after different concentrations of the small molecule compound T16 provided in Example 3 of the present invention acted on human T cells for 24 hours.

[0025] Figure 6 The expression level of GzmB after different concentrations of the small molecule compound T16 provided in Example 3 of the present invention acted on human T cells for 24 hours. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention. Some terms used in the present invention are listed below.

[0027] IFN-γ: It is an important immunomodulatory protein, mainly produced by activated T cells and natural killer cells. It has multiple biological functions, including immunomodulation, anti-virus, anti-tumor and immune balance regulation. IFN-γ can inhibit the proliferation of tumor cells, promote tumor cell apoptosis, and activate effector cells in the immune system to inhibit the growth of tumor cells.

[0028] IL-2: Interleukin-2 is a cytokine, also known as cell growth factor or T cell stimulating factor, which is mainly produced by activated T cells and has multiple biological activities, including promoting lymphocyte growth, proliferation and differentiation, and plays an important role in the body's immune response and anti-viral infection. IL-2 has a wide range of applications in clinical research and tumor treatment, and can be used to treat viral infections, immunodeficiency, and autoimmune system diseases, and also plays a certain role in tumor treatment research.

[0029] TNF-α: It is an important biologically active cytokine produced by activated macrophages, natural killer cells and T lymphocytes. TNF-α has the effect of killing tumors or inhibiting tumor proliferation both in vivo and in vitro.

[0030] GZMB: Granzyme B is a neutral serine protease present in the cytoplasmic granules of cytotoxic T cells and natural killer cells, playing a key role in the immune defense mechanism.

[0031] Autodock: It is a drug docking software based on the principles of molecular mechanics and computational chemistry. It predicts the binding ability and mode between drugs and proteins by simulating and calculating the interactions between molecules. The Autodock workflow includes preparing structure files, simulating interactions, searching for optimal solutions and calculating binding energy.

[0032] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0033] Example 1 Screening of small molecule binders to TIM-3.

[0034] Prepare protein and ligand files: Start the AutoDocktools molecular docking software, import the TIM-3 protein and process it according to the molecular docking operation procedures, including hydrogenation and energy optimization. Import the ligand molecules in the compound library Maybridge, add hydrogen, generate different ionization states and tautomers; generate grid files: use the Grid module (grid) to generate grid files, define the size and center of the grid (the parameters are the software default values); run the AutoDock Docking module for molecular docking (the parameters are the software default values), and screen out 10 small molecule compounds with docking scores less than -4.00, as shown in Table 1; among them, the compound with the highest absolute value of molecular docking score is compound T16, and its docking mode is as follows Figure 1 As shown, the small molecule compound T16 interacts with the 281st to 284th amino acid residues on the TIM-3 protein.

[0035] Table 1 Structures and docking results of the screened compounds Example 2: Surface ionosphere resonance (SPR) verification.

[0036] The 10 small molecule compounds screened in Example 1 were used to verify their binding to the TIM-3 protein using surface plasmon resonance.

[0037] Among them, the above 10 small molecule compounds were purchased from Merck with a purity greater than 98%. The above compounds were prepared into a mother solution of small molecule compounds with a concentration of 1M, and the solvent used for preparing the solution was dimethylsulfoxide (DMSO); Preparation of test sample solutions: The above-mentioned small molecule compound stock solutions were prepared with PBS-P buffer to prepare test sample solutions with concentrations of 20 µM, 40 µM, 80 µM, 160 µM and 320 µM; TIM-3 protein was purchased from KMD Biotechnology Co., Ltd.

[0038] The intracellular domain of TIM-3 protein was diluted to 50 µg / µl and fixed to the chip surface through the coupling procedure of the SPR instrument. The test sample solution was injected into the flow chamber of the SPR instrument, and the test sample flowed through the chip surface and interacted with the coupled ligand. The changes in the resonance angle were monitored in real time. These changes reflected the binding between the sample and the ligand. The changes in the resonance units (RU) were analyzed to determine the affinity of the intermolecular interaction. The verification results of the surface ion resonance are shown in Table 2 and Figure 2 As shown, among the 10 compounds initially screened, at a concentration of 80 μM, the RU values ​​of small molecule compounds T16, T17 and T18 were 7.5, 6.4 and 5.8, respectively. This result shows that the three small molecule compounds T16, T17 and T18 have strong binding ability with TIM-3 protein.

[0039] Example 3 Effects of small molecule compounds on T cell function.

[0040] T cells were isolated from blood samples from healthy individuals using conventional magnetic bead separation methods. The isolated T cells (2-4×10 6The cells were stimulated with anti-CD3 / CD28 antibodies, and the small molecule compound T16 was added at concentrations of 40 μM, 80 μM, and 160 μM for 24 h. The levels of IL-2, TNF-α, IFN-γ, and GzmB in the culture supernatant were determined by enzyme-linked immunosorbent assay. Test results such as Figures 3 to 6 As shown, compared with the control DMSO, the small molecule compound T16 solution with a concentration of 80 μM and 160 μM can significantly enhance the ability of T cells to secrete IFN-γ, TNF-α, IL-2 and GzmB.

[0041] in, Figures 3 to 6 ** means p value < 0.01, *** means p value < 0.001; ns is the abbreviation of not significant.

[0042] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A TIM-3 small molecule binder, characterized in that: It includes a small molecule compound that binds to the TIM-3 protein, and its structural formula is as follows: ； Among them, R1 is H or a halogen atom, R2 and R3 are each independently H or OH, and R4 is an alkyl group.

2. The TIM-3 small molecule binder according to claim 1, characterized in that The small molecule compound is selected from any one or more of the following structural formulas: , and 。 3. The TIM-3 small molecule binder according to claim 1, characterized in that It also includes at least one of a salt, a hydrate and a precursor compound of the small molecule compound.

4. The TIM-3 small molecule binder according to claim 1, characterized in that It also includes one or more of pharmaceutically acceptable adjuvants, excipients and carriers.

5. The TIM-3 small molecule binding agent according to claim 1, characterized in that Includes single preparations or compound preparations with small molecule compounds as active ingredients.

6. A method for screening a TIM-3 small molecule binder, characterized in that: For screening the TIM-3 small molecule binder according to any one of claims 1 to 5, comprising the following steps: S1. Screen the compounds in the compound library through the molecular docking platform to obtain compounds with a docking score lower than -4; S2. Use surface ion resonance technology to detect the binding force between the compound obtained in step S1 and the TIM-3 protein, and select small molecule compounds with a binding force greater than or equal to 5.8 as TIM-3 small molecule binders.

7. An application of a TIM-3 small molecule binder, characterized in that: The TIM-3 small molecule binder according to any one of claims 1 to 5, for use in the preparation of an immunomodulatory preparation.

8. The use of the TIM-3 small molecule binder according to claim 7, characterized in that: The immunomodulatory preparation includes at least one of an oral preparation, an injection preparation and a transdermal preparation.

9. The use of the TIM-3 small molecule binder according to claim 7, characterized in that: The immunomodulatory preparation is used to enhance the function of the body's effector T cells in secreting cytokines.

10. The use of the TIM-3 small molecule binder according to claim 7, characterized in that: The immunomodulatory preparation is used in tumor patients.

Images