BLT1 protein antagonist C858-0920 and application thereof

Abstract

The invention provides a BLT1 protein antagonist C858-0920 and application of the BLT1 protein antagonist C858-0920. It is found for the first time that C858-0920 and a BLT1 receptor have high affinity and can block combination of LTB4 and the BLT1 receptor, it is found that C858-0920 inhibits expression of LTB4-induced macrophage inflammation related genes Ccl4, Ccl2 and Spp1 and increases expression of vascular repair related genes Cxcl2, and it is shown that C858-0920 can relieve macrophage inflammation response and promote vascular repair, and then cardiac function repair after acute myocardial infarction is promoted. In addition, the C858-0920 is found to be capable of obviously improving the systolic function of the left ventricle, and the ejection fraction of the left ventricle is obviously improved compared with that of a PBS (Phosphate Buffer Solution) group; in addition, the FS value is also obviously increased; after the C858-0920 is subjected to intragastric administration, the size of the heart cavity of the left ventricle is reduced relative to that of a PBS group, LVESV and LVEDV are both reduced, statistical significance is achieved, and it is shown that C858-0920 promotes cardiac function repair after acute myocardial infarction.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a BLT1 protein antagonist, and specifically, to a BLT1 protein antagonist C858-0920 and an application thereof. Background Art

[0002] With the aging of the population and changes in lifestyle, the number of people suffering from cardiovascular diseases continues to increase. The mortality rate of cardiovascular diseases has exceeded that of cancer and other diseases. The mortality rate of cardiovascular diseases is high, and more than half of them die from acute myocardial infarction. Recently, the incidence of acute myocardial infarction has increased significantly and is showing a trend of younger age. In recent decades, the improvement of early intervention measures after acute myocardial infarction, especially the increase in timely reperfusion therapy, has significantly improved the survival rate of patients. Despite this, abnormal ventricular remodeling and heart failure after acute myocardial infarction remain a major challenge in clinical practice. Drugs developed globally for the treatment and improvement of ventricular remodeling after acute myocardial infarction have not been translated into clinical practice. Therefore, further exploration of treatment strategies for the prevention of heart failure after myocardial infarction has important scientific value and clinical significance.

[0003] The inflammatory response runs through all the pathological links of acute myocardial infarction injury and repair, and is the key mechanism causing myocardial injury after acute myocardial infarction. When the inflammatory pathway is activated, cytoplasmic phospholipase A2 (cPLA2) synthesizes arachidonic acid (AA) from membrane phospholipids, and then 5-LOX and 5-LOX activating protein (FLAP) convert AA into leukotriene B4 (LTB4). LTB4 is a lipid mediator with strong pro-inflammatory properties. In addition to acute inflammatory reactions, it is also associated with a variety of diseases, such as atherosclerosis, colon cancer, asthma, and pulmonary hypertension. LTB4 exerts biological effects through the corresponding G protein-coupled receptors on the target cell membrane, including BLT1 receptor and BLT2 receptor. BLT1 is a high-affinity receptor that is mainly expressed in leukocytes, such as neutrophils, eosinophils, and macrophages; BLT2 is a low-affinity receptor that is mainly expressed in the small intestine, liver, spleen, and leukocytes, and its function is still unclear. Therefore, screening antagonists targeting BLT1 protein and exploring its role in the inflammatory mechanism after myocardial infarction is one of the important topics in this field. Summary of the invention

[0004] In view of the deficiencies of the prior art, the object of the present invention is to provide a BLT1 protein antagonist C858-0920 and its application.

[0005] In the first aspect of the present invention, a use of a small molecule compound C858-0920 in the preparation of a BLT1 protein antagonist is provided, wherein the structural formula of the small molecule compound C858-0920 is:

[0006]

[0007] In the second aspect of the present invention, a use of a small molecule compound C858-0920 in the preparation of an anti-inflammatory drug is provided, wherein the structural formula of the small molecule compound C858-0920 is:

[0008]

[0009] Furthermore, the anti-inflammatory drug is a drug that reduces macrophage inflammation.

[0010] In the third aspect of the present invention, there is provided a use of a small molecule compound C858-0920 in the preparation of a drug for promoting vascular repair, wherein the structural formula of the small molecule compound C858-0920 is:

[0011]

[0012] In the fourth aspect of the present invention, there is provided a use of a small molecule compound C858-0920 in the preparation of a myocardial infarction prognosis drug, wherein the structural formula of the small molecule compound C858-0920 is:

[0013]

[0014] Furthermore, the myocardial infarction prognosis drug is a drug that promotes the repair of cardiac function after acute myocardial infarction.

[0015] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here.

[0016] Compared with the prior art, the present invention has the following advantages and progress:

[0017] The present invention provides a BLT1 protein antagonist C858-0920 and its application. The present invention first discovered that C858-0920 has a high affinity with the BLT1 receptor and can block the binding of LTB4 to the BLT1 receptor. At the same time, the present invention found through RT-qPCR results that C858-0920 inhibited LTB4-induced macrophage inflammation-related genes Ccl4, Ccl2, Spp1, and increased the expression of vascular repair-related gene Cxcl2, indicating that C858-0920 can reduce macrophage inflammatory response and promote vascular repair, thereby promoting cardiac function repair after acute myocardial infarction.

[0018] In addition, the present invention found that C858-0920 significantly improved the recovery of cardiac function after acute myocardial infarction through the results of mouse echocardiography. Specifically, the present invention found for the first time that C858-0920 can significantly improve the contractile function of the left ventricle, and the left ventricular ejection fraction was significantly higher than that of the PBS group; in addition, the FS value was also significantly increased; the left ventricular cavity size after C858-0920 gavage was smaller than that of the PBS group, and both LVESV and LVEDV decreased, which was statistically significant, indicating that C858-0920 promotes the recovery of cardiac function after acute myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0020] Figure 1 : Chemical structure formula of small molecule C858-0920.

[0021] Figure 2 : Molecular docking diagram of C858-0920 and BLT1.

[0022] Figure 3 :Possible interactions between target protein amino acids and small molecules during molecular docking.

[0023] Figure 4 : WT mouse BMDM cells were starved with serum-free DMEM for 24 h and then treated in different groups (PBS group, LTB4 group, C858-0920 group, LTB4+C858-092 group). The expression of inflammation-related genes was detected by qPCR (Means±SEM, *p<0.05; two-way ANOVA, followed by Bonferroni post hoc test for analysis, n=6).

[0024] Figure 5 : Effects of C858-0920 on cardiac function after acute myocardial infarction in mice (Means±SEM, *p<0.05; two-way ANOVA followed by Bonferroni post hoc test, n=6). DETAILED DESCRIPTION

[0025] The present invention provides a BLT1 protein antagonist C858-0920 and its application. The present invention is specifically described below in conjunction with the embodiments to facilitate further understanding of the present invention by those skilled in the art. However, the embodiments described below are only part of the embodiments of the present invention and should not be regarded as any form of limitation to the present invention. It should be pointed out that adjustments and improvements made by ordinary technicians in the field based on the concept of the present invention should be regarded as the scope of protection of the present invention. The specific technical operation steps and operators are not indicated in the embodiments, and they are all carried out according to the general technical conditions described in the literature of the field or the relevant product instructions.

[0026] Example

[0027] Part I Experimental Materials and Methods

[0028] 1. Construction of acute myocardial infarction (MI) model in mice:

[0029] 1. Mice were anesthetized with 1-2% isoflurane and then hair and skin were removed;

[0030] 2. Cut an opening of about 1 cm along the position where the heart beats with the largest amplitude, and insert a 4-0 surgical suture for later use;

[0032] 3. Bluntly separate the thorax, squeeze out the heart along the third and fourth intercostal spaces on the left side of the mouse chest, and suture it with 6-0 surgical sutures.

[0033] Ligation of the left anterior descending coronary artery;

[0034] 4. Replace the heart, expel the gas from the chest, and ligate the skin at the wound with 4-0 sutures;

[0035] 5. Stop anesthesia and put the mouse into a cage for further feeding after it regains consciousness.

[0036] 2. Mouse Cardiac Ultrasound

[0037] Transthoracic cardiac ultrasound examination of mice was performed using a Vevo 2100 high-resolution imaging system (Visualsonics, Toronto, ON, Canada) equipped with a 30 MHz probe, referring to the equipment operating manual. The specific steps are as follows:

[0038] 1. Anesthetize the mouse: Use a suitable anesthetic (such as isoflurane or pentobarbital) to fully anesthetize the mouse and ensure that it is in a pain-free and motionless state;

[0039] 2. Position the mouse: Place the mouse on a heating pad and adjust the position so that the chest is exposed to the ultrasound probe;

[0040] 3. Prepare the probe: Connect the 30MHz probe to the Vevo 2100 high-resolution imaging system and calibrate the probe to ensure good imaging quality;

[0041] 4. Apply gel: Apply a small drop of ultrasound gel on the appropriate position of the mouse's chest to provide an ultrasound transmission medium and reduce air gaps;

[0042] 5. Ultrasound imaging: Gently place the ultrasound probe on the chest area coated with ultrasound gel and fix it in a suitable position. Start the Vevo 2100 system for image acquisition;

[0043] 6. Obtain images: Use the real-time imaging function provided by the system to obtain cardiac images of mice;

[0044] 7. Data analysis: Import the collected electrocardiogram data into the analysis software to calculate and analyze cardiac function parameters;

[0045] 8. End of procedure: After completing image acquisition and data analysis, turn off the Vevo 2100 system, remove the mouse from the heating pad, and restore anesthesia.

[0046] 3. Isolation and Culture of Mouse Bone Marrow-derived Macrophages (BMDM)

[0047] 1. Remove leg bones from mice

[0048] After anesthesia, the mice were killed by dislocation. The mice were placed in a beaker containing sufficient 75% ethanol and soaked for disinfection for 5 minutes. The soaked animals were used to absorb excess alcohol. A small cut was made on the back of the mouse with scissors, and the skin was directly torn open to the joint of the mouse's calf by hand to remove the mouse's foot joint and skin. The hind limbs were removed along the greater trochanter of the mouse's thigh root with scissors, and the muscle tissue was removed and placed in a culture dish containing 75% ethanol and soaked for 5 minutes. A new culture dish containing 75% ethanol was replaced and moved to the clean bench.

[0049] 2. BMDM cell extraction and induction

[0050] Transfer the leg bones soaked in ethanol to cold PBS to wash off the ethanol on the surface of the tibia and femur. This process can be repeated 3 times. Separate the cleaned femur and tibia, and cut the two ends of the femur and tibia with scissors. Use a 1mL syringe to absorb the cold induction culture medium to blow the bone marrow out of the femur and tibia, and blow and wash repeatedly 3 times until no obvious red color is seen in the leg bones. Use a 5mL pipette to repeatedly blow the culture medium containing bone marrow cells to disperse the cell clumps, then use a 70μm cell filter to sieve the cells, transfer them to a 15mL centrifuge tube, centrifuge at 1500rpm / min for 5min, discard the supernatant, add red blood cell lysis buffer to resuspend, let stand for 5min, and then centrifuge at 1500rpm / min for 5min, discard the supernatant, resuspend with cold prepared bone marrow macrophage induction culture medium, and plate. The culture medium is not changed during cell culture. Half of the bone marrow macrophage induction culture medium is replaced after the third day of culture, and all the culture medium is replaced on the fifth day. It can be used for subsequent experiments on the seventh day.

[0051] 4. RT-qPCR detection of related gene expression

[0052] 1. Extraction of Total Cell RNA

[0053] (1) Aspirate the culture medium in the cell culture dish, add 500 μl Trizol reagent, pipette the cells, and transfer Trizol to a 1.5 ml Eppendorf tube;

[0054] (2) Add 100 μl of chloroform, shake vigorously to mix, and let stand at room temperature for 10 min;

[0055] (3) Centrifuge at 12,000 rpm for 10 min at 4°C and transfer the upper aqueous phase to a new Eppendorf tube;

[0056] (4) Add an equal volume of isopropanol, mix well, and let stand at room temperature for 10 min;

[0057] (5) Centrifuge at 12,000 rpm for 10 min at 4°C, discard the supernatant, and carefully remove the remaining liquid in the tube;

[0058] (6) Add 0.5 ml of 70% ethanol to wash the precipitate, make the precipitate completely float, and centrifuge at 8000 rpm for 7 min at 4°C;

[0059] (7) Discard the supernatant, air-dry the RNA, and add DEPC water;

[0060] (8) Melt in a 55°C metal bath for 5 min, place on ice for later use, and store at -80°C for long-term storage;

[0061] (9) Detect RNA concentration. RNA purity is expressed as A260 / A280, with an ideal value of 1.9-2.1;

[0062] 2. Reverse Transcription

[0063] Reaction system (20 μl):

[0064] Reagents volume <![CDATA[RNase-free ddH20]]> Make up to 15 μL 5xAll-in-one qRT SuperMix 4μL Enzyme Mix 1μL RNA 1 μg

[0065] Mix by gently pipetting, 37℃, 15min-85℃, 5s;

[0066] The cDNA of the reverse transcription product can be used immediately for qPCR reaction or stored at -20℃; C. Real-time fluorescence quantitative PCR

[0067] (1) Dilute the reverse transcribed cDNA solution 20 times;

[0068] (2) PCR primer sequences:

[0069]

[0070] (3) QPCR reaction system (20 μl):

[0071]

[0072]

[0073] (4) Reaction conditions

[0074] Reaction steps Number of cycles Reaction temperature Reaction time Pre-denaturation 1 95℃ 30s Cyclic reaction 40 95℃ 10s 60℃ 30s Melting curve 1 95℃ 15s 60℃ 60s 95℃ 15s

[0075] The relative gene expression was analyzed based on the CT value of the reaction.

[0076] 5. Data Statistical Analysis

[0077] Cell and molecular experiments were independently repeated at least three times. Animal experiments were repeated at least six times. Data from repeated experiments were averaged and expressed as mean ± standard error (SEM). Statistical analysis was performed using GraphPad Prism 5.0 software. Unpaired t-test was used to determine the differences between the two groups, and analysis of variance (ANOVA) was used for multiple group comparisons, followed by Bonferroni post hoc test to analyze the comparisons between groups. P < 0.05 was considered statistically significant.

[0078] Part II Experimental Results and Conclusions

[0079] 1. Molecular docking

[0080] The small molecule ligand (N-[(4-chlorophenyl)methyl]-2-(4-methylpiperidin-1-yl)-1,3-benzothiazole-6-carboxamide, C858-0920, CAS number: 906255-81-0) was obtained through the PubChem database (http: / / pubchem.ncbi.nlm.nih.gov / ). Figure 1 )’s 2D structure, input the 2D structure into Chem Office 20.0 software to create its 3D structure, and save it as a mol2 file. Then use the RCSB PDB database (http: / / www.rcsb.org / ) to screen protein targets with high resolution crystal structures as molecular docking receptors, and use PyMOL 2.6.0 software to perform dehydration, dephosphorylation and other operations on the protein, and save it as a PDB file. Molecular Operating Environment 2019 software was used to minimize the energy of the compounds, pre-treat the target proteins and search for active pockets. Finally, run MOE 2019 for molecular docking, and the number of operations was set to 50. The binding activity of the two was evaluated based on the binding energy, and the results were visualized using PyMOL 2.6.0 and Discovery studio 2019 software ( Figure 2 ).

[0081] According to the binding results, the number of atomic pairs with a distance less than 4A between BLT1 protein and C858-0920 small molecule is shown. Figure 3 The amino acids in the table may interact with small molecules (arranged from high to low in terms of the number of 4A atom pairs). Figure 3 The blue amino acids in the molecular formula are Figure 3 The amino acids marked in blue in the table are Figure 3 The red amino acids in the molecular formula are Figure 3 The amino acids marked in red in the table. It can be seen that from the perspective of molecular docking, C858-0920 has a high affinity with the BLT1 receptor protein ( Figure 2-3 ).

[0082] II. Biological function experiments of C858-0920 as a BLT1 protein antagonist

[0083] The C858-0920 small molecule (N-[(4-chlorophenyl)methyl]-2-(4-methylpiperidin-1-yl)-1,3-benzothiazole-6-carboxamide, CAS number: 906255-81-0) was purchased from ChemDiv under the trade name C858-0920.

[0084] The RT-qPCR experiment divided BMDM into four groups: PBS group, LTB4 group, C858-0920 group, and LTB4+C858-092 group. The PBS group and LTB4 group were treated with PBS or LTB4 for 6 hours, respectively. The C858-0920 group and LTB4+C858-092 group were treated with 100 nM C858-0920 for 1 hour, and then treated with PBS or LTB4 for 6 hours. According to the RT-qPCR results, the present invention found that C858-0920 inhibited LTB4-induced macrophage inflammation-related genes Ccl4, Ccl2, and Spp1, and increased the expression of vascular repair-related gene Cxcl2 ( Figure 4 ), indicating that C858-0920 can reduce macrophage inflammatory response and promote vascular repair, thereby promoting cardiac function repair after acute myocardial infarction.

[0085] At the same time, the present invention uses a mouse acute myocardial infarction model and finds, based on the results of echocardiography, that C858-0920 significantly improves cardiac function repair after acute myocardial infarction ( Figure 5 ). Specifically, the experiment divided the mice into two groups: MI+PBS group and MI+C858-0920 group (both groups were given PBS or C858-0920 (100 mg / kg / d) by gavage for 7 days before acute myocardial infarction surgery, and continued to be given until the 28th day after surgery). According to the results of echocardiography, it was found that C858-0920 could significantly improve the contractile function of the left ventricle, and the left ventricular ejection fraction was significantly higher than that of the PBS group ( Figure 5 ); In addition, the FS value also increased significantly ( Figure 5 ); the left ventricular cavity size after oral administration of C858-0920 was smaller than that of the PBS group, and both LVESV and LVEDV decreased ( Figure 5 The experimental results showed that C858-0920 promoted the recovery of cardiac function after acute myocardial infarction.

[0086] The above is only a preferred embodiment of the present invention and does not limit the present invention in any way. For those skilled in the art, any modification and change made to the above embodiment according to the technical essence of the present invention shall fall within the protection scope of the technical solution of the present invention.

Claims

1. Use of a small molecule compound C858-0920 in the preparation of a BLT1 protein antagonist, characterized in that: The structural formula of the small molecule compound C858-0920 is:

2. Use of a small molecule compound C858-0920 in the preparation of an anti-inflammatory drug, characterized in that: The structural formula of the small molecule compound C858-0920 is:

3. The use according to claim 2, characterized in that: The anti-inflammatory drug is a drug that reduces macrophage inflammation.

4. Use of a small molecule compound C858-0920 in the preparation of a drug for promoting vascular repair, characterized in that: The structural formula of the small molecule compound C858-0920 is:

5. Use of a small molecule compound C858-0920 in the preparation of a drug for the prognosis of myocardial infarction, characterized in that: The structural formula of the small molecule compound C858-0920 is:

6. The use according to claim 5, characterized in that: The myocardial infarction prognosis drug is a drug that promotes the repair of cardiac function after acute myocardial infarction.

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