CN110882262A - A drug for activation of myeloid cell trigger receptor 2 and its application - Google Patents

Abstract

The invention discloses the application of phosphorylated fingolimod in the preparation of a drug for the activation of myeloid cell trigger receptor 2, which is applied to the regulation of cell phagocytosis. The present invention discloses for the first time that phosphorylated fingolimod is an agonist of TREM2, and reveals that phosphorylated fingolimod enhances the phagocytic function of microglia by activating TREM2, and clarifies that phosphorylated fingolimod regulates microglia Novel mechanisms of cellular phagocytosis.

Description

A drug for activation of myeloid cell trigger receptor 2 and its application

technical field

The invention relates to the technical field of drug research and development, in particular to a drug for activation of myeloid cell trigger receptor 2 and its application.

Background technique

Microglia are innate immune effector cells in the central nervous system. In the normal development of the nervous system, microglia play functions such as rapid removal of apoptotic neurons, pruning of neuronal synapses, and secretion of cytokines to regulate neuron survival; in pathological conditions, microglia Cells are among the first immune cells to respond. For example, during brain damage such as ischemic stroke, a large number of neurons die, and the dead neurons release many dangerous molecules such as nucleic acids, proteins, and lipids, which in turn lead to inflammatory reactions. If these neurotoxic cell fragments are not obtained Effective removal will weaken the plasticity of neurons after injury, trigger secondary inflammation and aggravate the injury. Therefore, regulating the phagocytic function of microglia is one of the important ways to reduce nerve damage.

Triggering receptor expressed on myeliod cells 2 (TREM2) is a type I single transmembrane protein and a member of the immunoglobulin receptor family. TREM2 is highly expressed in macrophages, dendritic cells, osteoclasts and microglia. In the central nervous system, TREM2 is specifically expressed in microglia. Due to the lack of intracellular segment, TREM2 must realize signal transduction through its co-receptor 12kDa DNAX activating protein (DAP12), and finally play the functions of regulating phagocytosis, promoting cell growth, inhibiting apoptosis, and regulating inflammation. So far, although many substances have been found to bind to TREM2 to trigger downstream signals, such as phospholipids, nucleic acids, proteoglycans, heat shock protein 60 and apolipoprotein, etc., but the drugs that activate TREM2 have not yet been found.

Phosphorylated fingolimod (FTY720-Phosphate, FTY720-P) is the active form of fingolimod (FTY720) formed in vivo under the catalysis of sphingosine kinase 2 (SphK2), which is sphingosine-1-phosphate The structural analog of (sphingosine-1-phosphate, S1P) can activate S1PR1/3/4/5 and participate in many physiological functions mediated by it, such as cell survival, apoptosis, proliferation and other processes. The inventors newly discovered that FTY720 has the function of enhancing the phagocytosis of microglia, but it has not been reported whether it mediates the pro-phagocytic function by activating the phagocytosis-related receptors of microglia, such as myeloid trigger receptor 2 (TREM2).

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of phosphorylated fingolimod in the preparation of medicines for the activation of myeloid cell trigger receptor 2, so as to solve the above-mentioned problems in the prior art. Relationship between body 2 and identification of phosphorylated fingolimod as an activator of myeloid trigger receptor 2.

For achieving the above object, the present invention provides the following scheme:

The present invention provides the application of phosphorylated fingolimod in the preparation of a drug for the activation of myeloid cell trigger receptor 2, which is applied to the regulation of cell phagocytosis. The structural formula of the phosphorylated fingolimod (FTY720-P) is shown in formula I,

Preferably, it is used to enhance the phagocytic function of microglia.

Preferably, the phosphorylated fingolimod enhances the phagocytic function of microglia by activating myeloid trigger receptor 2.

Preferably, it is used to modulate inflammation. Specifically: Phosphorylation of fingolimod regulates inflammation by activating S1PR1/3/4/5.

The present invention discloses the following technical effects:

The present invention uses computer simulation to predict the possibility of human TREM2 (hTREM2) and FTY720-P binding, showing that hTREM2 binds to FTY720P through 47-arginine, 65-serine and 77-arginine (as shown in Figure 1) . It was further demonstrated that FTY720-P can directly bind to TREM2 by the method of micro thermal surge (MST), indicating that FTY720-P is a ligand of TREM2. In addition, when neurons are co-cultured with microglia, FTY720 treatment can enhance the phagocytosis of neuronal debris by microglia after oxygen-glucose deprivation-reperfusion injury. Attenuated, further from a functional point of view, FTY720-P plays a pro-phagocytic function by activating the TREM2 receptor. The invention discloses that FTY720-P is an agonist of TREM2, FTY720-P enhances the phagocytic function of microglia by acting on TREM2, and promotes the removal of cell debris in the case of pathological damage, and reveals that FTY720 regulates microglia cells New roles and new mechanics of features.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings required in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some of the present invention. In the embodiments, for those of ordinary skill in the art, other drawings can also be obtained according to these drawings without any creative effort.

Fig. 1 is the result diagram of TREM2 molecular docking of the present invention;

Fig. 2 is the result diagram of Micro Thermal Surge (MST) in Example 1 of the present invention;

Figure 3 shows the results of co-labeled immunofluorescence staining of microglia and neurons in the present invention; a: WB verifies the knockdown efficiency of TREM2 RNA interference (RNAi) on microglia; b: Iba1 Double-stained with NeuN immunofluorescence to reflect the phagocytosis of microglia; c: Statistics of the proportion of phagocytosed microglia;

Figure 4 is a graph showing the co-labeling results of Iba1 and CD68 immunofluorescence in the present invention; wherein, a: the result of immunofluorescence staining by Iba1 and CD68; b: the expression of CD68 was counted based on fluorescent staining;

Figure 5 is a graph showing the binding relationship between hTREM2 and FTY720 detected by the microcalorimetric surge (MST) method of the present invention.

Detailed ways

Various exemplary embodiments of the present invention will now be described in detail, which detailed description should not be construed as a limitation of the invention, but rather as a more detailed description of certain aspects, features, and embodiments of the invention.

It should be understood that the terms described in the present invention are only used to describe particular embodiments, and are not used to limit the present invention. Additionally, for numerical ranges in the present disclosure, it should be understood that each intervening value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value in that stated range is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Although only the preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference for the purpose of disclosing and describing the methods and/or materials in connection with which the documents are referred. In the event of conflict with any incorporated document, the content of this specification controls.

It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the present invention. The description and examples of the present application are only exemplary.

As used herein, "comprising," "including," "having," "containing," and the like, are open-ended terms, meaning including but not limited to.

Example 1

Microthermal surge method reveals the relationship between FTY720-P and TREM2

Using pET-24a(+) as a vector, construct various TREM2 protein expression plasmids marked by C-terminal His-tag. The sequences used for human-derived, rat-derived and mouse-derived TREM2 are respectively as SEQ ID NO: 1. Shown in SEQ ID NO: 2 and SEQ ID NO: 3.

Transform the recombinant plasmid into BL(DE3) E. coli: Take 100 μL of freshly prepared competent BL(DE3) cells, add 10 μL of the recombinant plasmid, flick and mix, and place on ice for 30 min; heat shock at 42°C for 80 s, then quickly transfer to ice In the bath, cool for 5 min; add 400 μL of LB medium at 37 °C, transfer to the incubator, and incubate at 37 °C for 1 h to recover the cells; take an appropriate amount of transformation product and spread it on LB plates containing kanamycin (15 g of agar, Peptone 10g, yeast extract 5g, NaCl 10g, water 1000mL), incubator at 37°C for 16h upside-down culture, pick a single colony for expansion and culture to 400mL culture medium, until the OD ₆₀₀ reaches about 0.8, add IPTG with a final concentration of 1mmoL/L Continue to culture for 8h to induce the expression of the target protein. Transfer the culture medium to a centrifuge tube, centrifuge at 4°C and 5000rpm for 5min, and collect the cells; resuspend the cells with pre-cooled tris buffer (Tris pH8.0 150mM, EDTA 10mM, NaCl 150mM), and centrifuge again at 5000rpm for 5min to collect Bacteria; use the His-Tag Protein Purification Kit to purify and collect the target protein, and use the Monolith His-Tag Labeling Kit to fluorescently label the His-tagged target protein, and detect the binding of TREM2 to S1P according to the instructions.

The results are shown in Figure 2. FTY720-P has binding ability to human, rat and mouse TREM2, and its KD values are 6.38 μM, 7.17 μM, and 72.60 μM, respectively (see Figure 2a, b for details). , c). Through molecular docking prediction, it was proved by the micro thermal surge (MST) method of this example that FTY720-P directly binds to TREM2, indicating that FTY720-P is a ligand of TREM2.

Example 2

Experimental grouping and administration

Rat primary neurons and microglia were extracted respectively, and the primary neurons were cultured in a 24-well plate (200,000 cells/well) with Neurobasal medium + 2% B27 + 1% double antibody, and the medium was half changed every other day. Cultured for one week until cells matured; primary microglia were cultured with 10% Gibco FBS+DMEM+1% double antibody, and the medium was changed every three days, and cultured for one week until cells matured.

After the cells matured, microglia were added to neurons for co-culture (20,000 cells/well), and the ratio of 1:10 was used for co-culture, and the Neurobasal medium without B27 was used: (10% Gibco FBS+DMEM+1% Double antibody)=3:1 culture medium, divided into normal group, normal administration group, normal knockdown group, knockdown administration group, and oxygen-glucose deprivation-reperfusion (OGD/R) injury at each time point (3h, 5h, 7h, 9h, 12h) of these four groups. The specific operation of oxygen glucose deprivation-reperfusion was as follows: 48h after knockdown transfection, OGD treatment was performed for 3h, normal medium or 10nM S1P treatment was changed during reperfusion, and the cells were washed with 0.01M PBS at each time point and fixed with paraformaldehyde for 1h.

Example 3

Immunofluorescence staining test

After the cells were fixed, they were washed 3 times with 0.01M PBS, discarded, and then blocked with blocking solution (containing 5% goat serum and 0.1% Triton X-100) for 1 h at room temperature; Incubate overnight at °C. After taking it out the next day, wash the primary antibody with 0.01M PBS, wash 3 times for 5 min each time, add the corresponding fluorescent secondary antibody (see Table 2 for antibody titer), incubate at room temperature for 1 h, and then wash with 0.01 M PBS for 3 times , 5 min each time; 5 μg/mL Hoechst solution was added dropwise to react in the dark for 20 min, washed 3 times with 0.01M PBS, 5 min each time, and photographed.

Table 1 Immunofluorescence staining with primary antibodies

Table 2 Immunofluorescence staining with secondary antibodies

The results are shown in Figure 3a for WB to verify the knockdown efficiency of TREM2 RNA interference (RNAi) on microglia; as shown in Figure 3b, double immunofluorescence staining with Iba1 and NeuN showed that the microglia were not damaged by OGD. Glial cells rarely had phagocytosis. In the control group, the clearance of neuronal debris by microglia was less until 3h after modeling, and increased significantly at 5h. The phagocytosis and clearance reached a peak at 7h, and then the clearance gradually weakened. As shown in Figure 3c, after S1P treatment, there was obvious clearance from 3h to 12h; after TREM2 knockdown, there was no obvious clearance of microglia in each group, indicating that FTY720- P promoted the phagocytosis of neuronal debris by microglia after oxygen-glucose deprivation-reperfusion injury, and the pro-phagocytosis of S1P was significantly reduced after TREM2 knockdown.

As shown in Figure 4, as a macrophage-specific lysosomal marker, CD68 can reflect the activation level and phagocytic ability of microglia. Figure 4a and b are the results of co-labeling of Ibal and CD68 by immunofluorescence. It can be seen from the figure that the expression of CD68 in microglia is very low in the absence of OGD injury. It reached a peak at 7h and decreased significantly at 9h; 3h after S1P treatment, there was a significant increase in CD68 expression, and there was still a high level of CD68 expression until 12h; after TREM2 knockdown, there was no significant increase in CD68 expression throughout the whole process; these results were all It indicated that FTY720-P enhanced the phagocytic clearance of neuronal debris by microglia, and TREM2 played an important role.

From the above results, it can be seen that when neurons are co-cultured with microglia, FTY720 treatment can enhance the phagocytosis of neuronal debris by microglia after oxygen-glucose deprivation-reperfusion injury, while knockdown of TREM2 significantly inhibits the expression of TREM2. Therefore, from a functional point of view, FTY720-P exerts a pro-phagocytic function by activating the TREM2 receptor.

It can be seen from Example 2 that the present invention is to directly administer FTY720. In order to exclude the direct effect of FTY720, the interaction relationship between TREM2 and FTY720 was further detected. The results are shown in Figure 5. There is no binding relationship between TREM2 and FTY720, indicating that the phosphorylation of FTY720 into FTY720-P can only bind to TREM2 for activation.

The above-mentioned embodiments are only to describe the preferred mode of the present invention, but not to limit the scope of the present invention. Without departing from the design spirit of the present invention, those of ordinary skill in the art can Variations and improvements should fall within the protection scope determined by the claims of the present invention.

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Claims (4)

1. The application of phosphorylated fingolimod in preparing a medicament for activating a myeloid cell trigger receptor 2 is characterized by being applied to regulating phagocytosis of cells.

2. Use of phosphorylated fingolimod in the manufacture of a medicament for the activation of myeloid cell triggering receptor 2 according to claim 1 for enhancing the phagocytic function of microglia.

3. Use of phosphorylated fingolimod in the manufacture of a medicament for activation of myeloid cell triggering receptor 2 according to claim 2 wherein phosphorylated fingolimod enhances microglial phagocytic function by activating myeloid cell triggering receptor 2.

4. Use of phosphorylated fingolimod in the manufacture of a medicament for the activation of myeloid cell triggering receptor 2 according to claim 1 for the modulation of inflammation.

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