A compound targeting TREM2 as an immunomodulator and its application.

Abstract

This invention discloses a compound targeting TREM2 as an immunomodulator and its applications. The compound represented by formula (I) exhibits phagocytic activity against various types of immune cells (microglia and macrophages). It is also demonstrated that it enhances the phagocytic activity of microglia and macrophages in various in vitro models. Furthermore, through methods such as microthermal surge, TREM2 knockdown, and the construction of a TREM2 / DAP12-CHO stable cell line, it is further shown that this novel compound enhances phagocytosis by targeting and binding to TREM2.

Description

Technical Field

[0001] This invention belongs to the field of small molecule compounds and relates to a compound that targets TREM2 as an immunomodulator and its application. Background Technology

[0002] Triggering receptor expressed on myeloid cells 2 (TREM2) is an immunoglobulin superfamily membrane receptor composed of an extracellular immunoglobulin-like domain, a transmembrane region, and a short cytoplasmic tail. TREM2 is primarily expressed on microglia and macrophages and can recognize a variety of ligands, such as lipoproteins, phospholipids, and certain pathogen-associated molecular patterns. After binding to ligands, TREM2 can modulate various cellular functions, including phagocytosis and immune responses.

[0003] Phagocytosis is a crucial mechanism for organisms to clear pathogens, cellular debris, and apoptotic cells. TREM2-mediated phagocytosis plays a vital role in various diseases. In neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), and Nasu-Hakola disease, TREM2 can initiate microglia to phagocytose and clear the accumulation of abnormal substances such as β-amyloid plaques, α-synuclein, and myelin fragments, thereby protecting neurons by reducing neurotoxicity and improving cellular metabolism. In acute brain injuries such as stroke and traumatic brain injury, TREM2 participates in the clearance of damaged neurons, cellular debris, and myelin fragments by microglia, reducing neuroinflammatory responses. Activation of TREM2 within peripheral macrophages mediates the clearance of damaged cells, apoptotic cells, immune complexes, bacteria, viruses, and tumors, thereby playing a regulatory role in autoimmune diseases, chronic inflammatory diseases, infectious diseases, and tumors. Based on the crucial regulatory role of TREM2-mediated phagocytosis in cellular immunity, the development of TREM2-targeting modulators holds promise for providing new strategies for treating immune-related diseases. Summary of the Invention

[0004] The purpose of this invention is to provide a compound that acts as a regulator targeting TREM2 to enhance the phagocytic activity of immune cells.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] The compound represented by formula (Ⅰ):

[0007]

[0008] The use of the compound in the preparation of drugs or in vitro reagents that upregulate the phagocytic activity of microglia and / or macrophages.

[0009] As a preferred embodiment of the present invention, the compound is capable of enhancing the phagocytic activity of microglia and / or macrophages.

[0010] As a preferred embodiment of the present invention, the compound can enhance the phagocytic activity of microglia and macrophages in various in vitro models.

[0011] As a preferred embodiment of the present invention, the compound's effect in promoting microglia phagocytosis depends on its binding to Trem2.

[0012] As a preferred embodiment of the present invention, the compound promotes phagocytosis in TREM2 / DAP12-CHO stable cells.

[0013] Beneficial effects:

[0014] This invention discloses a class of compounds and demonstrates their phagocytic-enhancing effects on various types of immune cells (primary microglia, microglial cell lines, and macrophages). Furthermore, through methods such as microthermal surge, TREM2 knockdown, and the construction of a TREM2 / DAP12-CHO stable cell line, it is further shown that these compounds enhance phagocytosis by targeting and binding to TREM2. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 The figure shows the effects of these compounds on the viability of microglia BV2, macrophage IBMDM, and primary microglia.

[0017] Figure 2 The figure shows the results of this type of compound promoting phagocytosis in the microglia BV2 cell line.

[0018] Figure 3 The figure shows the results of this type of compound promoting phagocytosis in the IBMDM macrophage cell line.

[0019] Figure 4 The figure shows the results of this type of compound promoting phagocytosis in primary microglia.

[0020] Figure 5 The figure shows the results of these compounds promoting microglia phagocytosis in an in vitro model of neuroinflammation.

[0021] Figure 6 The figure shows the results of these compounds promoting microglia phagocytosis in an in vitro model of Alzheimer's disease.

[0022] Figure 7 This figure shows the results of these compounds promoting phagocytosis in microglia in an in vitro model of multiple sclerosis.

[0023] Figure 8 The graph shows the binding results of this type of compound with mTREM2 and hTREM2, respectively.

[0024] Figure 9 The figure shows the results of this type of compound promoting phagocytosis in primary microglia after TREM2 knockdown.

[0025] Figure 10 The figure shows the results of this type of compound promoting phagocytosis in the TREM2 / DAP12-CHO stable cell line. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] Example 1: Extraction of primary microglia

[0028] Purchase 1-2 day old suckling mice. After disinfecting the mice with 75% alcohol, remove their brains. Wash the brains in pre-cooled DMEM medium, then remove the meninges and blood vessels using ophthalmic forceps. Discard the DMEM medium and use curved scissors to mince the brain tissue for easier digestion. Mix 0.25% trypsin with PBS at a 1:1 ratio and add the brain tissue. Incubate at 37°C for 15 minutes, shaking the culture dish every 5 minutes to ensure thorough digestion. Add complete culture medium to stop digestion. Collect the tissue suspension in a 50mL centrifuge tube and centrifuge at 1000g for 5 minutes at room temperature. Discard the supernatant. Add complete culture medium and pipette the tissue to release cells. Pipe approximately 18 times and allow the suspension to stand. Allow any undispersed brain tissue to settle to the bottom of the tube. Collect the supernatant single-cell suspension in another centrifuge tube. Add fresh complete culture medium to the brain tissue and repeat the above steps, pipetting until no obvious brain tissue blocks remain, minimizing cell damage. The collected single-cell suspension was filtered through a 400-mesh sterile sieve. After filtration, the cells were counted and seeded into culture flasks pre-coated with L-poly-L-lysine (0.1 mg / mL) and placed in a cell culture incubator at 37°C. Microglia were cultured in a different medium on the second day after extraction, and thereafter every 3 days. After one week of culture, microglia were isolated by tapping for experimental use.

[0029] Example 2: Cell viability assay

[0030] Different types of immune cells (microglia BV2, macrophages IBMDM, and primary microglia) were seeded into 96-well plates and cultured for 12 hours. Cells were then treated with different concentrations of compound #17 for 24 hours. After 24 hours, 100 μL of DMEM medium containing 10 μL of CCK-8 solution was added to each well, and the plates were incubated for 1 hour. After 1 hour, the OD values ​​of the 96-well plates were measured using a microplate reader with an excitation light of 450 nm to observe the effect of this compound on the viability of different immune cells.

[0031] like Figure 1 As shown, with increasing concentrations of these compounds, the viability of three immune cell lines—BV2 microglia, IBMDM macrophages, and primary microglia—remained largely unchanged. Furthermore, at the highest concentration of 10 μM, none of these compounds exhibited toxicity to BV2 microglia, IBMDM macrophages, or primary microglia. These results indicate that within the set concentration range, these compounds do not affect the viability of BV2 microglia, IBMDM macrophages, or primary microglia.

[0032] Example 3: Phagocytosis Experiment

[0033] Each cell type was seeded at its designated seeding density in 24-well plates. After cell adhesion, the cells were washed twice with PBS to remove residual serum. Cytoplasmic staining was performed according to the manufacturer's instructions without affecting the cell morphology. 1 μM Cell Tracker solution was added to each well. TM Green dye was used to culture cells in a 37°C cell culture incubator for 20 min. After the incubation, residual dye was washed off with PBS, and the cells were cultured in serum-free medium. Cells were treated with 1 μL of red zymosan bioparticles per 100 μL of medium, and different concentrations of this TREM2 immunomodulator were also administered. Phagocytosis images were collected after 6 h to observe the effect of this TREM2 immunomodulator on the phagocytic capacity of three types of immune cells. The ratio of the number of cells with phagocytic function in the field of view to the total number of cells was used as the phagocytic rate, and the phagocytosis images were quantitatively analyzed.

[0034] like Figure 2 As shown, compared with the untreated control group, all four concentrations of this compound (0.1 μM, 0.5 μM, 1 μM, and 5 μM) significantly enhanced the phagocytic function of the microglia BV2 cell line. Furthermore, the phagocytic rate mediated by the 0.5 μM concentration of this compound was significantly higher than that mediated by the 0.1 μM concentration, with a statistically significant difference. Figure 3 As shown, compared with the untreated control group, both 1 μM and 10 μM concentrations of these compounds promoted enhanced phagocytic function in the IBMDM macrophage cell line. Figure 4 As shown, compared with the untreated control group, concentrations of these compounds at 1 μM and 10 μM significantly enhanced the phagocytic function of primary microglia. The results indicate that the newly synthesized compounds significantly enhance the phagocytic function of three immune cell lines: primary microglia, BV2 microglia, and IBMDM macrophages.

[0035] Example 4: LPS-induced in vitro inflammation model

[0036] After microglia were seeded into 24-well plates and adhered, they were stimulated with LPS (1 μg / mL) for 12 h, and then treated with different concentrations of such compounds while simultaneously being given pHrodo to conduct phagocytosis experiments.

[0037] like Figure 5 As shown, compared with the LPS group, concentrations of these compounds at 1 μM and 10 μM significantly enhanced the phagocytic function of microglia.

[0038] Example 5: Ex vivo model of aplastic anemia

[0039] After microglia were seeded and adhered to 24-well plates, they were stimulated with FAM-Aβ (500 nM) and then treated with different concentrations of this compound, while simultaneously receiving pHrodo for phagocytosis experiments. In this model, the phagocytosed material appeared as sheets, and quantitative analysis was performed directly based on the fluorescence intensity of Aβ ingested within individual cells.

[0040] like Figure 6 As shown, concentrations of these compounds at 1 μM and 10 μM significantly enhanced the phagocytic function of microglia after Aβ treatment.

[0041] Example 6: Ex vivo model of multiple sclerosis

[0042] After microglia were seeded and adhered to 24-well plates, they were stimulated with myelin (20 μg / ml), then treated with different concentrations of this compound, and simultaneously administered pHrodo for phagocytosis experiments. In this model, the phagocytosed material appeared as sheets, and quantitative analysis was performed directly based on the fluorescence intensity of myelin absorbed within individual cells.

[0043] like Figure 7 As shown, a 10 μM concentration of this compound significantly enhances the phagocytic function of microglia after myelin treatment. Figures 5-8 The results indicate that these compounds can significantly enhance the phagocytic function of microglia in in vitro models of various nervous system diseases, further clarifying the immunomodulatory effect of these compounds in promoting phagocytosis.

[0044] Example 7: Preparation of recombinant TREM2 protein

[0045] Human and murine pET-24a(+)-TREM2 plasmids were constructed (gene sequences are shown in Table 1). All plasmids were individually transformed into *E. coli* BL21 competent cells and cultured at 37°C with shaking until the OD600 reached approximately 0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium at a final concentration of 1 mmol / L, and the cells were cultured for another 8 h to induce TREM2 expression. After expression, the cells were collected by centrifugation and washed with pre-cooled Tris buffer. The cells were resuspended in non-denaturing lysis buffer and sonicated for 20 min to disrupt the cells (ultrasonic parameters set to 2 s sonication followed by 2 s intervals). The supernatant was collected by centrifugation for protein purification. The supernatant was purified into TREM2 protein using a His-tag Purification Resin kit: the supernatant was mixed with His-tag Purification Resin after equilibration with lysis buffer and shaken slowly on a shaker for 1 hour to fully bind the tag protein; the His-tag Purification Resin was packed into a column, and after removing unbound protein with washing buffer, TREM2 protein was eluted with non-denaturing elution buffer and collected, further concentrated and purified by ultrafiltration, and BCA protein quantification was performed.

[0046] Table 1. Sequences of human and murine pET-24a(+)-TREM2 plasmids

[0047]

[0048]

[0049] Example 8: Micro-heat surge

[0050] TREM2 protein with his tag was fluorescently labeled with RED-tris-NTA2nd Generation dye. The target molecule was serially diluted from the highest concentration to a total of 16 concentrations. The ligand of each concentration was mixed with an equal volume of fluorescent TREM2 recombinant protein and aspirated into Monolith NT.115 capillaries. The thermal migration in each capillary was detected using a Monolith NT.115 microthermophoresis instrument. The binding of different TREM2 recombinant proteins to the target molecule was analyzed using MO.AffinityAnalysis software.

[0051] like Figure 8 As shown, these compounds can directly bind to both human recombinant TREM2 protein and mouse recombinant TREM2 protein, respectively, and their K... D The values ​​were 804.41±416.08 nM and 1.88±6.51 μM, respectively. The results indicate that these compounds have a strong binding affinity to TREM2.

[0052] Example 9: Knockdown of TREM2 receptor in primary microglia

[0053] Primary microglia were seeded at a density of 600,000 cells per well in 6-well plates. After cell attachment on the second day, siRNA transfection was performed as follows: 5 μl / well Messenger MAX TM The reagent was diluted with 125 μl of Opti-MEM solution, gently mixed, and incubated at room temperature for 10 minutes. Three siRNA concentrations were prepared, diluted with Opti-MEM solution to a final concentration of 125 μl, gently mixed, and added to the previously incubated solution. After further gentle mixing, the mixture was incubated at room temperature for 5 minutes. The cell culture medium was replaced with 1.75 ml of Opti-MEM solution, and the prepared transfection reagent was slowly added dropwise to each well, mixing constantly. The plate was incubated at 37°C in a 5% CO2 incubator. Knockout efficiency was assessed one day after transfection. The concentration that did not damage the cells to achieve the maximum knockout rate was selected for subsequent experimental procedures.

[0054] Table 2. siRNA sequences of TREM2

[0055]

[0056] like Figure 9 As shown, in primary microglia treated with TREM2 siRNA, the phagocytic capacity of the Trem2 KD+Cmpound group showed no significant change compared to the Trem2KD group, indicating that these compounds do not affect the phagocytic function of primary microglia after TREM2 knockdown. Furthermore, compared to the Vehicle+Cmpound group, the phagocytic capacity of the Trem2 KD+Cmpound group was significantly reduced, indicating that knocking out TREM2 in primary microglia reverses the enhancement of phagocytic function promoted by these compounds. This suggests that these compounds exert their phagocytic enhancement effect on primary microglia through the TREM2 receptor.

[0057] Example 10: Construction of a stable TREM2 / DAP12-CHO cell line

[0058] The signal peptide CD8 leader sequence, along with the chimeric mRNA sequence of the extracellular and transmembrane regions of TREM2 and the intracellular region of DAP12, was loaded into the pLenti-CMV-GFP-Puro vector to construct the plasmid of the pLenti-CMV-CD8A-mTREM2 lentiviral vector (plasmid sequence shown in Table 3, device location details are provided in Table 3). Figure 10a) Culturing CHO cells and treating them with different concentrations of G418, a death curve was established to determine the optimal screening concentration of G418. The above plasmid was transfected into CHO cells using Lipofectamine 3000. After 24 hours, adherent cells were passaged, and after another 24 hours, resistance was expressed. At this time, the optimal concentration of G418 was used for selection. Cells were observed daily, and the medium containing the same concentration of G418 was changed in a timely manner. After one week of culture, the cells were digested and dispersed, and seeded into 96-well plates at a density of only one cell per well. Wells with only a single cell were selected for further culture, expanded, and passaged into 24-well plates, and then into 6-well plates. Some cells were isolated for phagocytosis experiments.

[0059] Table 3. Sequence of pLenti-CMV-CD8A-mTREM2 chimeric protein particles

[0060]

[0061]

[0062] like Figure 10 As shown, compared with normal CHO cells, the phagocytic function of CHO cells treated with these compounds showed no significant change, and both types of cells exhibited almost no phagocytic ability. Compared with normal CHO cells, the phagocytic activity of TREM2 / DAP12-CHO stable cells was significantly enhanced. However, the phagocytic activity of TREM2 / DAP12-CHO stable cells treated with these compounds was significantly enhanced compared with untreated TREM2 / DAP12-CHO stable cells. This further indicates that the TREM2 receptor is a key target for these compounds to enhance the phagocytic activity of primary microglia.

Claims

1. The compound represented by formula (Ⅰ): 。 2. The use of the compound of claim 1 in the preparation of a drug or in vitro reagent that upregulates the phagocytic activity of microglia and / or macrophages.

3. The application according to claim 2, characterized in that, The compound of claim 1 can enhance the phagocytic activity of microglia and / or macrophages.

4. The application according to claim 2, characterized in that, The use of the compound of claim 1 in the preparation of reagents for enhancing the phagocytic activity of microglia and / or macrophages in an in vitro model.

5. The application according to claim 2, characterized in that, The use of the compound of claim 1 in the preparation of a reagent that promotes microglia phagocytosis in an in vitro model of neuroinflammation.

6. The application according to claim 2, characterized in that, The use of the compound of claim 1 in the preparation of a reagent that promotes phagocytosis by microglia in an in vitro model of Alzheimer's disease.

7. The application according to claim 2, characterized in that, The use of the compound of claim 1 in the preparation of a reagent that promotes phagocytosis by microglia in an in vitro model of multiple sclerosis.

8. The application according to claim 2, characterized in that, The effect of the compound of claim 1 in promoting microglia phagocytosis depends on its binding to Trem2.

9. The application according to claim 8, characterized in that, The compound of claim 1 promotes phagocytosis in TREM2 / DAP12-CHO stable cells.

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