Targeted TREM2 compound as immunomodulator and application thereof

Abstract

The invention discloses a compound targeting TREM2 as an immunomodulator and application of the compound. The compound as shown in the formula (I) has a phagocytosis promoting effect on various types of immune cells (microglial cells and macrophages). And the phagocytosis of microglial cells and macrophages under various in-vitro models can be enhanced. In addition, through micro thermal surging, TREM2 knock-down, TREM2 / DAP12-CHO stably transfected cell strain construction and other methods, it is further shown that the novel structure compound plays a role in enhancing cell phagocytosis through targeted combination with TREM2. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of small molecule compounds and relates to a compound targeting TREM2 as an immunomodulator and an application thereof. Background Art

[0002] Triggering receptor expressed on myeloid cells 2 (TREM2) is an immunoglobulin superfamily membrane receptor, which consists of an extracellular immunoglobulin-like domain, a transmembrane region, and a short cytoplasmic tail. TREM2 is mainly expressed on microglia and macrophages, and it can recognize a variety of ligands, such as lipoproteins, phospholipids, and certain pathogen-associated molecular patterns. After binding to ligands, TREM2 can regulate multiple functions of cells, such as phagocytosis and immune response.

[0003] Phagocytosis is an important mechanism for organisms to remove pathogens, cell debris and apoptotic cells. TREM2-mediated phagocytosis plays an important role in many diseases. In neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), multiple sclerosis (MS), Nasu-Hakola disease, etc., TREM2 can activate the phagocytosis of microglia to remove the accumulation of abnormal substances such as β-amyloid plaques, α-synuclein and myelin fragments, so as to reduce neurotoxicity, improve cell metabolism and play a protective role on neurons. In acute brain injury diseases such as stroke and brain trauma, TREM2 participates in the removal of damaged neurons, cell debris and myelin fragments by microglia, reducing neuroinflammatory response. Activation of TREM2 in peripheral macrophages can mediate the clearance of damaged cells, apoptotic cells, immune complexes, bacteria, viruses and tumors by macrophages, thereby playing a regulatory role in autoimmune diseases, chronic inflammatory diseases, infectious diseases and tumor diseases. Based on the key regulatory role of TREM2-mediated phagocytic function on cellular immune function, the development of regulators targeting TREM2 is expected to provide a new strategy for the treatment of immune-related diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a compound as a regulator targeting TREM2 to enhance the phagocytic effect of immune cells.

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

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

[0007]

[0008] The compound is used in preparing a drug or an in vitro reagent for up-regulating the phagocytic function of microglia and / or macrophages.

[0009] As a preferred embodiment of the present invention, the compound can enhance the phagocytic function of microglia and / or macrophages.

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

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

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

[0013] Beneficial effects:

[0014] The present invention discloses a class of compounds, and confirms that they have a phagocytic effect on various types of immune cells (primary microglia, microglia cell lines and macrophages). In addition, through methods such as microcalorimetry, knocking down TREM2 and constructing a TREM2 / DAP12-CHO stable cell line, it is further shown that such compounds enhance cell phagocytosis by targeting TREM2. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0016] Figure 1 The graphs show the effects of this type of compounds on the viability of microglial cell line BV2 cells, macrophage cell line IBMDM cells and primary microglial cells.

[0017] Figure 2 This figure shows the results of this type of compound promoting the phagocytosis of microglial cell line BV2 cells.

[0018] Figure 3 This figure shows the results of this type of compounds promoting the phagocytosis of the macrophage cell line IBMDM cells.

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

[0020] Figure 5 This figure shows the results of this type of compound promoting the phagocytosis of microglia in an in vitro model of neuroinflammation.

[0021] Figure 6 This figure shows the results of this type of compound promoting the phagocytosis of microglia in an in vitro model of Alzheimer's disease.

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

[0023] Figure 8 These are the results of the binding of this type of compounds to mTREM2 and hTREM2 respectively.

[0024] Fig. 9 This figure shows the results of this type of compounds promoting the phagocytosis of primary microglia after TREM2 knockdown.

[0025] Fig.10 This figure shows the results of this type of compounds promoting the phagocytosis of TREM2 / DAP12-CHO stable cell lines. DETAILED DESCRIPTION

[0026] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1: Extraction of primary microglia

[0028] Purchase newborn 1-2 day old suckling mice, disinfect them with 75% alcohol, remove the brain, soak them in pre-cooled DMEM culture medium for washing, remove the meninges and blood vessels with ophthalmic forceps, discard the DMEM culture medium, and cut the brain tissue into pieces for easy digestion. Mix 0.25% trypsin with PBS at a ratio of 1:1 and add it to the brain tissue. Place it in a 37℃ cell culture incubator for digestion for 15 minutes, and shake the culture dish every 5 minutes to fully digest the tissue. Add complete culture medium to terminate digestion, collect the tissue suspension in a 50mL centrifuge tube, centrifuge it at room temperature at 1000g for 5 minutes, discard the supernatant, add complete culture medium to blow the tissue to release the cells, blow about 18 times and let the suspension stand, wait for the brain tissue that has not been blown away to sink to the bottom of the tube, aspirate the upper single cell suspension and collect it in another centrifuge tube, add new complete culture medium to the brain tissue, repeat the above operation and blow it several times until there is no obvious brain tissue block to reduce damage to the cells. The collected single cell suspension was filtered with a 400-mesh sterile sieve, counted and inoculated into a culture bottle pre-coated with L-polylysine (0.1 mg / mL) and placed in a 37°C cell culture incubator for culture. The microglia were replaced with a medium on the second day of extraction, and then every 3 days. After one week of culture, microglia were separated by beating method for experiment.

[0029] Example 2: Cell viability detection

[0030] Different types of immune cells (microglial cell line BV2, macrophage cell line IBMDM and primary microglia) were inoculated in 96-well plates, and the culture plates were cultured in an incubator for 12 hours. The cells were treated with different concentrations of 17# compound for 24 hours. After 24 hours, 100 μL DMEM culture medium containing 10 μL CCK-8 solution was added to each well, and the culture plates were placed in an incubator for incubation for 1 hour. After 1 hour, the 96-well plates were placed on a microplate reader with an excitation light of 450 nm for OD value detection to observe the effect of such compounds on the activity of different immune cells.

[0031] like Figure 1 As shown, with the increase of the concentration of such compounds, the viability of three immune cells, microglial cell line BV2 cells, macrophage cell line IBMDM cells and primary microglial cells, did not change significantly. And at the highest concentration of 10μM, there was no toxic effect on microglial cell line BV2 cells, macrophage cell line IBMDM cells and primary microglial cells. The results show that within the set concentration range, such compounds do not affect the viability of microglial cell line BV2 cells, macrophage cell line IBMDM cells and primary microglial cells.

[0032] Example 3: Phagocytosis experiment

[0033] Each cell type was seeded in a 24-well plate at its respective seeding density. After the cells adhered to the plate, they were washed twice with PBS to remove the residual serum. The cells were stained for cytoplasm according to the instructions without affecting the state of the cells themselves. Cell Tracker was added to the cells in each well at a concentration of 1 μM. TM Green dye, cultured in a 37°C cell culture incubator for 20 minutes, washed with PBS to remove residual dye, replaced with serum-free culture medium for culture, and treated cells at a ratio of 1 μL red phagocytic dye (pHrodoRed zymosan bioparticles) per 100 μL culture medium, and treated with different concentrations of such TREM2 immunomodulators. After 6 hours, phagocytic images were collected to observe the effects of such TREM2 immunomodulators on the phagocytic ability of the three immune cells. The ratio of the number of cells with phagocytic function to the total number of cells in the field of view was used as the phagocytic rate of the cells, and the phagocytic images were quantitatively analyzed.

[0034] like Figure 2 As shown, compared with the untreated control group, the four concentrations of 0.1μM, 0.5μM, 1μM, and 5μM of these compounds can significantly enhance the phagocytic function of the microglial cell line BV2 cells. And the phagocytic rate mediated by the 0.5μM concentration of these compounds is significantly higher than that of the 0.1μM concentration of these compounds, with a statistically significant difference. Figure 3 As shown, compared with the untreated control group, 1μM and 10μM concentrations of these compounds can promote the enhancement of the phagocytic function of the macrophage cell line IBMDM cells. Figure 4 As shown, compared with the untreated control group, 1μM and 10μM concentrations of this type of compound can significantly enhance the phagocytic function of primary microglia. The results show that the newly synthesized compounds have the ability to significantly enhance the phagocytic function of three immune cells: primary microglia, microglial cell line BV2 cells, and macrophage cell line IBMDM cells.

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

[0036] After microglia were seeded into 24-well plates and adhered to the wall, they were stimulated with LPS (1 μg / mL) for 12 h, and then treated with different concentrations of such compounds, and pHrodo was given at the same time to carry out phagocytosis experiments.

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

[0038] Example 5: AD in vitro model

[0039] After microglia were seeded into 24-well plates and attached to the wall, they were stimulated with FAM-Aβ (500nM) and treated with different concentrations of such compounds, and pHrodo was given at the same time to conduct phagocytosis experiments. In this model, the phagocytosis appeared in sheets, and quantitative analysis was performed directly based on the fluorescence intensity of engulfed Aβ in a single cell.

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

[0041] Example 6: In vitro model of multiple sclerosis

[0042] After microglia were seeded into 24-well plates and attached to the wall, they were stimulated with myelin (20 μg / ml), and then treated with different concentrations of this compound, and pHrodo was given at the same time to conduct phagocytosis experiments. In this model, the phagocytosis appeared in sheets, and quantitative analysis was performed directly based on the fluorescence intensity of engulfed myelin in a single cell.

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

[0044] Example 7: Preparation of recombinant TREM2 protein

[0045] Humanized and mouse pET-24a(+)-TREM2 plasmids were constructed (see Table 1 for gene sequences). All the above plasmids were separately transferred into E. coli BL21 competent cells and cultured at 37°C with shaking until OD600 reached about 0.8. Isopropyl-β-D-thiogalactoside (IPTG) was added to the culture medium at a final concentration of 1 mmol / L and cultured for 8 hours to induce TREM2 expression. After the expression, the cells were collected by centrifugation and washed with pre-cooled Tris buffer. The cells were resuspended in non-denaturing lysis buffer, and the cells were broken by ultrasonic treatment for 20 minutes (ultrasonic parameters were set to ultrasonic 2s, intermittent 2s), and the supernatant was collected by centrifugation for protein purification. The supernatant was purified using a his-tag protein purification kit to purify TREM2 protein: the supernatant was mixed with the His-tag Purification Resin equilibrated with the lysate, and the mixture was shaken slowly on a shaker for 1 hour to fully bind the tagged protein; the His-tag Purification Resin was loaded onto the column, unbound proteins were removed by washing solution, and the TREM2 protein was eluted and collected with a non-denaturing eluent, and further concentrated and purified by ultrafiltration through an ultrafiltration tube, and the BCA protein was quantified.

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

[0047]

[0048]

[0049] Example 8: Microthermal surge

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

[0051] like Figure 8 As shown in Figure 2, these compounds can directly bind to human recombinant TREM2 protein and mouse recombinant TREM2 protein, respectively, and their K D The values ​​were 804.41±416.08nM and 1.88±6.51μM, respectively. The results showed that this type of compound has a strong binding ability with TREM2.

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

[0053] Primary microglia were inoculated into 6-well plates at a density of 600,000 cells per well. The next day, after the cells adhered to the wall, the following siRNA transfection operation was performed. Take 5 μl / well Messenger MAX TM Reagent, dilute with 125μl Opti-MEM solution, gently mix and incubate at room temperature for 10 minutes; set the concentration of three siRNAs, dilute with Opti-MEM solution to 125μl system, gently mix and add to the above incubated solution, continue to gently mix and incubate at room temperature for 5 minutes. Replace the cell culture medium with 1.75ml Opti-MEM solution, and then slowly drip the prepared transfection reagent into each well, mixing the solution in the plate while dripping. Place in a 37℃ constant temperature incubator containing 5% CO2 and culture. Detect the knockout efficiency one day after transfection. Select the concentration that does not damage the maximum knockout rate of cells for subsequent experimental operations.

[0054] Table 2 siRNA sequences of TREM2

[0055]

[0056] like Fig. 9 As shown in the figure, in primary microglia treated with TREM2 siRNA, there was no significant change in the phagocytic ability of cells in the Trem2 KD+Cmpound group compared with the Trem2KD group, indicating that this type of compound does not affect the phagocytic function of primary microglia after TREM2 knockdown. In addition, compared with the Vehicle+Cmpound group, the phagocytic ability of cells in the Trem2 KD+Cmpound group was significantly reduced, indicating that knocking out TREM2 in primary microglia would reverse the enhancement of the phagocytic function of primary microglia by this type of compound. This shows that this type of compound enhances the phagocytic effect of primary microglia through the TREM2 receptor.

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

[0058] The signal peptide CD8 leader sequence, the chimeric mRNA sequence of the extracellular and transmembrane regions of TREM2 and the intracellular region of DAP12 were loaded into the pLenti-CMV-GFP-Puro vector to construct the pLenti-CMV-CD8A-mTREM2 lentiviral vector plasmid (the plasmid sequence is shown in Table 3, and the device location is shown in Fig.10a). CHO cells were cultured and treated with different concentrations of G418, and a death curve was established to determine the optimal screening concentration of G418. The above plasmid was transfected into CHO cells using Lipofectamine 3000, and the adherent cells were passaged after 24 hours. After another 24 hours, the cells expressed resistance, and then the optimal concentration of G418 was used for screening. The cells were observed daily and the culture medium containing the same concentration of G418 was replaced in time. After one week of culture, the cells were digested and blown away, and seeded in a 96-well plate at a density of only one cell per well. The wells with only a single cell were screened and continued to be cultured. After amplification, they were passaged in a 24-well plate, and then passaged to a 6-well plate, and some cells were separated for phagocytosis experiments.

[0059] Table 3 pLenti-CMV-CD8A-mTREM2 chimeric protein plasmid sequence

[0060]

[0061]

[0062] like Fig.10 As shown in the figure, compared with normal CHO cells, there was no significant change in the phagocytic function of CHO cells treated with such compounds, and both cells had almost no phagocytic ability. Compared with normal CHO cells, the phagocytic activity of TREM2 / DAP12-CHO stable cells has been significantly enhanced. However, the phagocytic activity of TREM2 / DAP12-CHO stable cells treated with such compounds was significantly enhanced compared with that of untreated TREM2 / DAP12-CHO stable cells. This further indicates that the TREM2 receptor is the key target for such compounds to enhance the phagocytic activity of primary microglia.

Claims

1. The compound represented by formula (I):

2. Use of the compound according to claim 1 in the preparation of a drug or in vitro reagent for upregulating the phagocytic function of microglia and / or macrophages.

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

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

5. The effect according to claim 2, characterized in that Use of the compound of claim 1 in the preparation of an agent for promoting microglial phagocytosis in an in vitro model of neuroinflammation.

6. The effect according to claim 2, characterized in that Use of the compound of claim 1 in the preparation of an agent for promoting microglial phagocytosis in an in vitro model of Alzheimer's disease.

7. The effect according to claim 2, characterized in that Use of the compound of claim 1 in the preparation of an agent for promoting microglial phagocytosis in an in vitro model of multiple sclerosis.

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

9. The effect according to claim 8, characterized in that The compound described in claim 1 has the ability to promote phagocytosis of TREM2 / DAP12-CHO stable cells.

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