WO2022134733A1 - Use of compound capable of inhibiting interaction of coronavirus spike protein with ace2 - Google Patents
Use of compound capable of inhibiting interaction of coronavirus spike protein with ace2 Download PDFInfo
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- WO2022134733A1 WO2022134733A1 PCT/CN2021/122540 CN2021122540W WO2022134733A1 WO 2022134733 A1 WO2022134733 A1 WO 2022134733A1 CN 2021122540 W CN2021122540 W CN 2021122540W WO 2022134733 A1 WO2022134733 A1 WO 2022134733A1
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Definitions
- the present invention relates to pharmaceutical use, in particular to a compound capable of inhibiting the interaction between coronavirus Spike protein and ACE2.
- the novel coronavirus pneumonia caused by SARS-CoV-2 was named by WHO as COVID-2019. So far, there is no effective way to treat this infectious disease. Therefore, there is a need to produce vaccines or antibody drugs to fight the disease or to screen new drugs for the treatment of coronavirus disease.
- SARS-CoV-2 utilizes the homotrimeric spike (S) glycoprotein to bind to angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD), which is further processed by human proteases Proteolytic activation, thereby mediating cell infection.
- SARS-CoV-2 spike protein and the human ACE2 receptor is currently considered a key therapeutic strategy for coronavirus infection.
- the purpose of the present invention is to provide the use of a compound capable of inhibiting the interaction between coronavirus Spike protein and ACE2.
- the present invention provides the use of a compound capable of inhibiting the interaction between coronavirus Spike protein and ACE2 in the preparation of a medicine for treating and/or preventing SARS-CoV-2 novel coronavirus infection.
- the structure of the compound is such as formula I:
- R1 or R2 groups are respectively selected from H, F, Cl, Br, I, CF 3 , CF 3 O, CN, CH 3 O, CH 3 CH 2 O, CH 3 CH 2 CH 2 O, NH 2 , CH2NH2 , CH3 , CH3CH2 , CH3CH2CH2 or OH .
- the median inhibitory concentrations of compound 1, compound 2 and compound 3 were 0.96 ⁇ M, 0.10 ⁇ M and 0.16 ⁇ M, respectively.
- the compound can specifically bind to the RBD region of Spike protein.
- the compound can inhibit the binding of the coronavirus Spike protein to the ACE2 protein, and can also promote the dissociation of the Spike-ACE2 complex.
- the pharmaceutical dosage form is an oral dosage form or a non-oral dosage form.
- the oral dosage form is tablet, powder, granule, capsule, emulsion or syrup.
- non-oral dosage form is an injection.
- the preparation method of the compound capable of inhibiting the interaction between coronavirus Spike protein and ACE2 comprises the following steps:
- step (3) Dissolving the product in step (2) in distilled water, ultrafiltration and centrifugation for 0.5 ⁇ 2h, taking the lower layer solution, concentrating under reduced pressure, recovering the solvent, separating and collecting by high performance liquid chromatography to obtain compounds 1 and 2 respectively , 3.
- the high-efficiency liquid phase separation conditions in step (3) include: chromatographic column specifications are C18, 5 ⁇ m, 4.6 ⁇ 250 mm Grace visionHT; 0.1% aqueous formic acid-acetonitrile gradient elution with a volume ratio of 95:5 to 65:35 is used to detect The wavelength is 240-260nm, and the flow rate is 1-2mL/min.
- coronavirus spike protein helps the virus to recognize and enter the target cell, it is a key protein for virus invasion. Therefore, targeting the interaction between coronavirus Spike protein and human ACE2 receptor protein is a key therapeutic strategy for coronavirus infection, and small molecule inhibitors of protein-protein interaction between Spike and ACE2 can be applied to target Spike mediators. Prevention and/or treatment of induced coronavirus infection.
- the inventors used an activity-oriented separation strategy to isolate a class of compounds with kynurenic acid as the parent nucleus from natural products that can inhibit the interaction between Spike protein and ACE2 protein with IC 50 ⁇ 1 ⁇ M, and can promote the Spike-ACE2 complex. dissociate.
- the compounds can effectively inhibit the invasion of the novel coronavirus SARS-CoV-2 pseudovirus at the cellular level, with IC 50 ⁇ 2 ⁇ M.
- the compounds can specifically bind to the RBD region of the Spike protein with K D ⁇ 6 ⁇ M, and it is found that these compounds can be used as effective coronavirus invasion inhibitors.
- the present invention includes a pharmaceutical composition
- a pharmaceutical composition comprising a compound represented by formula I or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer or racemate thereof as an active ingredient, and a pharmaceutically acceptable Accepted excipients.
- the pharmaceutically acceptable excipient refers to any diluent, adjuvant or carrier that can be used in the pharmaceutical field.
- the compounds of the present invention may be used in combination with other active ingredients as long as they do not produce other adverse effects.
- the compound of the present invention can bind to the RBD region on the spike protein of SARS-CoV-2 that binds to the ACE2 receptor, effectively inhibits the interaction between the RBD region of SARS-CoV-2 and ACE2, prevents the virus from entering cells, and at the same time can Promoting the dissociation of the Spike-ACE2 complex can be used to prepare a drug for treating and/or preventing SARS-CoV-2 novel coronavirus infection.
- Figure 1 shows the dose-dependent blocking of the interaction between SARS-CoV-2 RBD and ACE2 protein by compound 1 and the measurement results of the half-inhibitory concentration
- Figure 2 shows the dose-dependent blocking of the interaction between SARS-CoV-2 RBD and ACE2 protein by compound 2 and the results of the half-inhibitory concentration assay
- Figure 3 shows the dose-dependent blocking of the interaction between SARS-CoV-2 RBD and ACE2 protein by compound 3 and the results of the half-inhibitory concentration assay
- Figure 4 is the assay result of the compound of the present invention promoting the dissociation activity of Spike and ACE2 complex, (wherein: **P ⁇ 0.01, ***P ⁇ 0.001 vs. solvent control group);
- Figure 5 shows the results of affinity determination of compound 1 binding to SARS-CoV-2 RBD
- Figure 6 shows the results of affinity determination of compound 2 binding to SARS-CoV-2 RBD
- Figure 7 is the result of affinity determination of compound 3 binding to SARS-CoV-2 RBD
- Figure 8 shows the results of the toxicity assay of recombinant human ACE2 protein on 293T-ACE2 cells
- Figure 9 shows the results of the toxicity assay of compound 1 on 293T (293T-ACE2) cells overexpressing the ACE2 receptor;
- Figure 10 shows the results of the toxicity assay of compound 2 on 293T-ACE2 cells
- Figure 11 shows the results of the toxicity assay of compound 3 on 293T-ACE2 cells
- Figure 12 is a fluorescence microscope observation of compounds 1, 2, and 3 inhibiting SARS-CoV-2 novel coronavirus pseudovirus from invading 293T-ACE2 cells;
- Figure 13 shows the dose-dependent inhibition of the SARS-CoV-2 novel coronavirus pseudovirus from invading 293T-ACE2 cells by recombinant human ACE2 protein and the assay results of the half-inhibitory concentration;
- Figure 14 shows the dose-dependent inhibition of SARS-CoV-2 novel coronavirus pseudovirus from invading 293T-ACE2 cells and the assay results of the half-inhibitory concentration
- Figure 15 shows the dose-dependent inhibition of the SARS-CoV-2 novel coronavirus pseudovirus from invading 293T-ACE2 cells and the half-inhibitory concentration assay results of compound 2;
- Figure 16 shows the dose-dependent inhibition of the SARS-CoV-2 novel coronavirus pseudovirus from invading 293T-ACE2 cells and the half-inhibitory concentration assay results of compound 3;
- Figure 17 is a model control group that only added the new coronavirus S protein pseudovirus
- Figure 18 is a positive control group added with a mixture of novel coronavirus S protein pseudovirus and 180nM ACE2 recombinant protein;
- Figure 19 is the experimental group added with a mixture of novel coronavirus S protein pseudovirus and 5 ⁇ M compound 2;
- Figure 20 shows the dose-dependent inhibition of SARS-CoV-2 novel coronavirus pseudovirus intrusion into Calu-3 cells and the half-inhibitory concentration determination results of compound 2 of the present invention.
- step (3) Dissolving the product in step (2) in distilled water, ultrafiltration and centrifugation for 0.5h, taking the lower layer solution, concentrating under reduced pressure, recovering the solvent, and separating by high performance liquid chromatography to obtain compound 1.
- the high-performance liquid phase separation conditions described in the above step (3) include: the chromatographic column specification is C18, 5 ⁇ m, 4.6 ⁇ 250 mm GracevisionHT; the mobile phase is 0.1% formic acid aqueous solution (A) and acetonitrile (B), and the gradient elution program is 0- 10min, 5%A; 10–50min, 5%–20%A; 50–60min, 20%–25%A; 60–75min, 25–35%A; 75–76min, 35%–5%A; 76 –85min, 5%A.
- the flow rate was 1.0ml/min
- the detection wavelength was 254nm
- the column temperature was kept at 35°C
- the retention time on the high-performance liquid phase was about 8.4min.
- step (3) Dissolving the product in step (2) in distilled water, ultrafiltration and centrifugation for 0.5h, taking the lower layer solution, concentrating under reduced pressure, recovering the solvent, and separating by high performance liquid chromatography to obtain compound 2.
- the high-performance liquid phase separation conditions described in the above step (3) include: the chromatographic column specification is C18, 5 ⁇ m, 4.6 ⁇ 250mm Grace visionHT; the mobile phase is 0.1% formic acid aqueous solution (A) and acetonitrile (B), and the gradient elution program is 0-10min, 5%A; 10–50min, 5%–20%A; 50–60min, 20%–25%A; 60–75min, 25–35%A; 75–76min, 35%–5%A ;76–85min, 5%A.
- the flow rate was 1.0ml/min
- the detection wavelength was 254nm
- the column temperature was kept at 35°C
- the retention time on the high-performance liquid phase was about 13.9min.
- step (3) Dissolving the product in step (2) in distilled water, ultrafiltration and centrifugation for 0.5 h, taking the lower layer solution, concentrating under reduced pressure, recovering the solvent, and separating by high performance liquid chromatography to obtain compound 3.
- the high-performance liquid phase separation conditions described in the above step (3) include: the chromatographic column specification is C18, 5 ⁇ m, 4.6 ⁇ 250mm Grace visionHT; the mobile phase is 0.1% formic acid aqueous solution (A) and acetonitrile (B), and the gradient elution program is 0-10min, 5%A; 10–50min, 5%–20%A; 50–60min, 20%–25%A; 60–75min, 25–35%A; 75–76min, 35%–5%A ;76–85min, 5%A.
- the flow rate was 1.0ml/min
- the detection wavelength was 254nm
- the column temperature was kept at 35°C
- the retention time on the high-performance liquid phase was about 24.7min.
- the compound of the present invention inhibits the interaction between the RBD region of the SARS-CoV-2 novel coronavirus Spike protein and the ACE2 receptor.
- chromogenic solution sodium hydrogen phosphate-citrate buffer containing 0.1 mg/ml TMB and 0.004% hydrogen peroxide, pH 5.5
- reaction stop solution 1M sulfuric acid
- the compound of the present invention promotes the dissociation of the SARS-CoV-2 novel coronavirus Spike protein RBD region and the ACE2 receptor complex.
- chromogenic solution sodium hydrogen phosphate-citrate buffer containing 0.1 mg/ml TMB and 0.004% hydrogen peroxide, pH 5.5
- reaction stop solution 1M sulfuric acid
- the absorbance was read at 450 nm using a microplate reader.
- Raw data were analyzed using GraphPad Prism 6.0 software. Measured values are expressed as the mean ⁇ standard error of the mean of three independent experiments. Between-group analysis was performed using one-way ANOVA, and p ⁇ 0.05 was considered statistically significant.
- the compound of the present invention interacts with the RBD region of the SARS-CoV-2 novel coronavirus Spike protein.
- SPR sensing technology uses the principle of surface plasmon resonance.
- the SPR resonance signal is very sensitive to the change of the refractive index of the sample material on the sensor chip surface, so the material information of the sample can be obtained by analyzing the SPR resonance image. It has extremely important applications in the field of biosensing such as the detection of protein-protein interactions.
- the experiments were performed at 25°C using a BIAcore T200 instrument with a mobile phase of phosphate buffered saline (pH 7.4).
- the SARS-CoV-2 RBD recombinant protein was diluted with 10 mmol/L sodium acetate buffer (pH values were 5.5, 5.0, 4.5 and 4.0) to a final concentration of 20 ⁇ g/ml, and the pH value was screened by manual injection method. Connect the pH value with the maximum value for subsequent experiments.
- the SARS-CoV-2 RBD recombinant protein was coupled to the CM5 chip by amino coupling method.
- SARS-CoV-2 was diluted with 10 mmol/L sodium acetate buffer with the best pH value obtained from the above screening.
- CoV-2 RBD recombinant protein to a final concentration of 20 ⁇ g/ml.
- the chip surface was activated with a mixture of 0.2 mol/L EDC and 50 mmol/L NHS in a ratio of 1:1, injected at a flow rate of 10 ⁇ l/min, and injected with SARS-CoV-2 RBD recombinant protein solution after continuous injection for 7 min.
- the sample was injected with 1 mol/L ethanolamine hydrochloric acid (pH 8.5) blocking solution for 7 min to block the activated chip surface, and the blocked blank channel was used as a negative control for the determination.
- the compound of the present invention with the highest concentration of 25 ⁇ M was selected as the ligand, and the ligand was injected into the immobilized SARS-CoV-2 RBD recombinant protein biosensor chip in two-fold serial dilution.
- a 1:1 binding model was used to assess binding kinetics. KD values were calculated by BIAcore T200 analysis software using the kinetic model.
- the compound of the present invention inhibits the invasion of 293T cells (293T-ACE2) overexpressing the ACE2 receptor by the pseudovirus of the S protein of the SARS-CoV-2 novel coronavirus.
- the new coronavirus S protein pseudovirus invades cells.
- the SARS-CoV-2 novel coronavirus S protein pseudovirus contains a green fluorescent protein reporter gene.
- the expression of green fluorescent protein can be observed under a fluorescence microscope, which reflects the number of cells invaded by the virus.
- the binding of the new coronavirus S protein to the ACE2 receptor is a key step for the virus to invade cells, so 293T cells (293T-ACE2) overexpressing the ACE2 receptor were selected to simulate virus invasion.
- the MTT method was used to detect the effect of each compound on cell viability and whether it had cytotoxicity.
- 293T-ACE2 cells were evenly plated into well plates, and DMEM medium containing 10% fetal bovine serum was used for cell culture. After culturing for 24 hours at 37°C and 5% CO2, various compounds were added to each well. , and incubated for another 24 hours. 293T-ACE2 cells were then placed in 100 ⁇ l of 10% FBS-DMEM containing 500 ⁇ g/ml MTT. After 4 hours of incubation at 37°C, 150 ⁇ l of dimethyl sulfoxide was added to each well to dissolve the crystals. Absorbance values were measured at 570 nm using a microplate reader.
- Compounds 1-3 can prevent the entry of the new coronavirus S protein pseudovirus into 293T-ACE2 cells.
- compounds 1-3 and the positive control hACE2 recombinant protein had no effect on the viability of 293T-ACE2 cells within a certain concentration range, indicating that these compounds are safe.
- Figure 12 it can be clearly observed under the fluorescence microscope that compared with the 293T cells that do not express the ACE2 receptor, the 293T-ACE2 cells in the model group express a large amount of green fluorescent protein, indicating that ACE2 helps the new coronavirus S protein pseudovirus to invade key receptors in cells.
- the compound of the present invention inhibits the invasion of SARS-CoV-2 novel coronavirus S protein pseudovirus into human lung glandular epithelial cells (Calu-3).
- the new coronavirus S protein pseudovirus invades cells.
- the SARS-CoV-2 novel coronavirus S protein pseudovirus contains a green fluorescent protein reporter gene. When the virus invades cells, the expression of green fluorescent protein can be observed under a fluorescence microscope, which reflects the number of cells invaded by the virus.
- Human lung gland epithelial cells (Calu-3) were evenly plated into the well plate, and the cells were cultured in MEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 , and the cells were grown to 50%. Start experimenting. The same concentration of SARS-CoV-2 novel coronavirus S protein pseudovirus was mixed with different concentrations of the test compounds, incubated at 37°C for 1 h, and then added to each well.
- Figures 17, 18, and 19 are fluorescence microscope observations of compound 2 inhibiting SARS-CoV-2 novel coronavirus pseudovirus from invading human lung glandular epithelial cells (Calu-3). As shown in the figure, compound 2 can prevent The novel coronavirus S protein pseudovirus enters Calu-3 cells.
- Figure 17 is a model control group with only the new coronavirus S protein pseudovirus added.
- Figure 18 is a positive control group added with a mixture of novel coronavirus S protein pseudovirus and 180nM ACE2 recombinant protein.
- Figure 19 shows the experimental group added with a mixture of novel coronavirus S protein pseudovirus and 5 ⁇ M compound 2.
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Abstract
Provided are a use of a compound capable of inhibiting the interaction of coronavirus Spike protein with ACE2, and a use of the compound in the preparation of a drug for treating and/or preventing SARS-CoV-2 infection. The compound can not only inhibit the interaction between coronavirus Spike protein and ACE2 protein, with IC50<1 μM, but also promote the dissociation of Spike-ACE2 complex. In addition, at the cellular level, the compound can effectively inhibit the invasion of pseudovirus of SARS-CoV-2, with IC50<2 μM. The compound can specifically bind to an RBD region of Spike protein, with KD<6 μM, which shows that the compound has a very positive effect on the preparation of medicaments for the treatment and/or prevention of coronavirus infection.
Description
本发明涉及药物用途,特别涉及能够抑制冠状病毒Spike蛋白与ACE2相互作用的化合物。The present invention relates to pharmaceutical use, in particular to a compound capable of inhibiting the interaction between coronavirus Spike protein and ACE2.
一种病毒SARS-CoV-2是一种新型冠状病毒,由SARS-CoV-2引起新型冠状病毒肺炎被WHO命名为COVID-2019。到目前为止,还没有有效的方法来治疗这种传染病。因此,需要生产疫苗或抗体药物来对抗这种疾病或筛选用于治疗冠状病毒疾病的新药物。A virus, SARS-CoV-2, is a new type of coronavirus. The novel coronavirus pneumonia caused by SARS-CoV-2 was named by WHO as COVID-2019. So far, there is no effective way to treat this infectious disease. Therefore, there is a need to produce vaccines or antibody drugs to fight the disease or to screen new drugs for the treatment of coronavirus disease.
冠状病毒的刺突(Spike)蛋白有助于病毒识别并进入靶细胞。许多研究小组表明,SARS-CoV-2利用同源三聚体刺突(S)糖蛋白通过其受体结合结构域(RBD)与血管紧张素转化酶2(ACE2)结合,进一步被人类蛋白酶进行蛋白水解激活,从而介导细胞感染。靶向SARS-CoV-2突刺蛋白和人类ACE2受体之间的相互作用目前被认为是一种关键的冠状病毒感染治疗策略。现有研究表明,针对SARS-CoV-2的中和抗体或者可溶性重组人ACE2受体可以有效的阻止Spike-ACE2蛋白-蛋白相互作用,减少病毒对于人肺上皮细胞的入侵,从而达到预防或者治疗COVID-2019的目的。然而与其他的蛋白质疗法相似,抗体或蛋白药物的应用还存在许多问题,比如免疫原性、口服生物利用度差、产品均一性差、生产和储存过程中不稳定等问题,蛋白类药物的使用仍然有很大的限制。发展小分子的蛋白-蛋白相互作用抑制剂更加具有挑战性,但是同时它的很多特性利于药物开发,比如优良的组织渗透性,低免疫原性,高稳定性,易于化学合成生产等优势。因此,寻找Spike蛋白与ACE2蛋白相互作用的阻断剂对于制备治疗和/或预防冠状病毒感染药物具有重要意义。The spike protein of coronaviruses helps the virus to recognize and enter target cells. Numerous research groups have shown that SARS-CoV-2 utilizes the homotrimeric spike (S) glycoprotein to bind to angiotensin-converting enzyme 2 (ACE2) through its receptor-binding domain (RBD), which is further processed by human proteases Proteolytic activation, thereby mediating cell infection. Targeting the interaction between the SARS-CoV-2 spike protein and the human ACE2 receptor is currently considered a key therapeutic strategy for coronavirus infection. Existing studies have shown that neutralizing antibodies against SARS-CoV-2 or soluble recombinant human ACE2 receptors can effectively prevent Spike-ACE2 protein-protein interactions and reduce virus invasion of human lung epithelial cells, thereby achieving prevention or treatment. Purpose of COVID-2019. However, similar to other protein therapies, there are still many problems in the application of antibody or protein drugs, such as immunogenicity, poor oral bioavailability, poor product uniformity, instability during production and storage, etc. The use of protein drugs is still There are big restrictions. The development of small-molecule protein-protein interaction inhibitors is more challenging, but at the same time, many of its properties are beneficial to drug development, such as excellent tissue permeability, low immunogenicity, high stability, and easy chemical synthesis and production. Therefore, it is of great significance to find a blocking agent for the interaction between Spike protein and ACE2 protein for the preparation of drugs for the treatment and/or prevention of coronavirus infection.
发明内容SUMMARY OF THE INVENTION
发明目的:本发明目的是提供能够抑制冠状病毒Spike蛋白与ACE2相互作用的化合物的用途。Purpose of the invention: The purpose of the present invention is to provide the use of a compound capable of inhibiting the interaction between coronavirus Spike protein and ACE2.
技术方案:本发明提供一种能够抑制冠状病毒Spike蛋白与ACE2相互作用的化合物在制备治疗和/或预防SARS-CoV-2新型冠状病毒感染的药物中的用途,化合物的结构如式I:Technical scheme: The present invention provides the use of a compound capable of inhibiting the interaction between coronavirus Spike protein and ACE2 in the preparation of a medicine for treating and/or preventing SARS-CoV-2 novel coronavirus infection. The structure of the compound is such as formula I:
式IFormula I
其中,R1或R2基团分别选自H、F、Cl、Br、I、CF
3、CF
3O、CN、CH
3O、CH
3CH
2O、CH
3CH
2CH
2O、NH
2、CH
2NH
2、CH
3、CH
3CH
2、CH
3CH
2CH
2或OH。
Wherein, R1 or R2 groups are respectively selected from H, F, Cl, Br, I, CF 3 , CF 3 O, CN, CH 3 O, CH 3 CH 2 O, CH 3 CH 2 CH 2 O, NH 2 , CH2NH2 , CH3 , CH3CH2 , CH3CH2CH2 or OH .
进一步地,化合物为如下几种:Further, the compounds are as follows:
化合物1、化合物2、化合物3的半数抑制浓度分别为0.96μM、0.10μM和0.16μM。The median inhibitory concentrations of compound 1, compound 2 and compound 3 were 0.96 μM, 0.10 μM and 0.16 μM, respectively.
进一步地,化合物能特异性的结合在Spike蛋白的RBD区域。Further, the compound can specifically bind to the RBD region of Spike protein.
进一步地,化合物可以抑制冠状病毒Spike蛋白与ACE2蛋白的结合,也可以促使Spike-ACE2复合物的解离。Further, the compound can inhibit the binding of the coronavirus Spike protein to the ACE2 protein, and can also promote the dissociation of the Spike-ACE2 complex.
进一步地,所述药物剂型为口服给药剂型或非口服给药剂型。Further, the pharmaceutical dosage form is an oral dosage form or a non-oral dosage form.
进一步地,所述的口服给药剂型为片剂、散剂、颗粒剂、胶囊剂、乳剂或糖浆剂。Further, the oral dosage form is tablet, powder, granule, capsule, emulsion or syrup.
进一步地,所述的非口服给药剂型是注射剂。Further, the non-oral dosage form is an injection.
所述的能够抑制冠状病毒Spike蛋白与ACE2相互作用的化合物的制备方法,包括如下步骤:The preparation method of the compound capable of inhibiting the interaction between coronavirus Spike protein and ACE2 comprises the following steps:
(1)取麻黄,40~50℃烘干,粉碎后过24~100目筛,取筛下粉末备用;(1) get ephedra, dry at 40~50 ℃, pass through 24~100 mesh sieves after pulverizing, get the powder under the sieve for subsequent use;
(2)向麻黄粉末中加入其重量10~100倍的重量百分比浓度为70%~100%的乙醇进行超声波辅助提取,提取温度为25~60℃,提取时间为0.5~2h,超声频率为30~40KHz,过滤,去除溶剂,得到浸膏备用;(2) Add 10 to 100 times its weight of ethanol with a concentration of 70% to 100% by weight to the ephedra powder to carry out ultrasonic-assisted extraction, the extraction temperature is 25 to 60°C, the extraction time is 0.5 to 2h, and the ultrasonic frequency is 30 ~40KHz, filter, remove the solvent, and obtain the extract for use;
(3)将步骤(2)中的产物溶解于蒸馏水中,超滤离心0.5~2h,取下层溶液,减压浓缩、回收溶剂,经过高效液相色谱分离、分步收集分别得到化合物1、2、3。(3) Dissolving the product in step (2) in distilled water, ultrafiltration and centrifugation for 0.5~2h, taking the lower layer solution, concentrating under reduced pressure, recovering the solvent, separating and collecting by high performance liquid chromatography to obtain compounds 1 and 2 respectively , 3.
步骤(3)所述高效液相分离条件包括:色谱柱规格为C18,5μm,4.6×250mm Grace visionHT;用体积比为95:5到65:35的0.1%甲酸水溶液-乙腈梯度洗脱,检测波长为240-260nm,流速1-2mL/min。The high-efficiency liquid phase separation conditions in step (3) include: chromatographic column specifications are C18, 5 μm, 4.6×250 mm Grace visionHT; 0.1% aqueous formic acid-acetonitrile gradient elution with a volume ratio of 95:5 to 65:35 is used to detect The wavelength is 240-260nm, and the flow rate is 1-2mL/min.
由于冠状病毒刺突蛋白有助于病毒识别并进入靶细胞,是病毒入侵的关键蛋白。 因此,靶向冠状病毒Spike蛋白和人类ACE2受体蛋白之间的相互作用是一种关键的冠状病毒感染治疗策略,Spike与ACE2之间蛋白质-蛋白质相互作用小分子抑制剂可应用于针对Spike介导的冠状病毒感染的预防和/或治疗。发明人采用活性为导向的分离策略从天然产物中分离了以kynurenic acid为母核的一类化合物能够抑制Spike蛋白与ACE2蛋白的相互作用,IC
50<1μM,同时能够促使Spike-ACE2复合物的解离。在细胞水平上可以有效的抑制新型冠状病毒SARS-CoV-2假病毒入侵,IC
50<2μM。所述化合物能特异性的结合在Spike蛋白的RBD区域,K
D<6μM,发现该类化合物可作为有效的冠状病毒入侵抑制剂。
Since the coronavirus spike protein helps the virus to recognize and enter the target cell, it is a key protein for virus invasion. Therefore, targeting the interaction between coronavirus Spike protein and human ACE2 receptor protein is a key therapeutic strategy for coronavirus infection, and small molecule inhibitors of protein-protein interaction between Spike and ACE2 can be applied to target Spike mediators. Prevention and/or treatment of induced coronavirus infection. The inventors used an activity-oriented separation strategy to isolate a class of compounds with kynurenic acid as the parent nucleus from natural products that can inhibit the interaction between Spike protein and ACE2 protein with IC 50 <1μM, and can promote the Spike-ACE2 complex. dissociate. It can effectively inhibit the invasion of the novel coronavirus SARS-CoV-2 pseudovirus at the cellular level, with IC 50 <2μM. The compounds can specifically bind to the RBD region of the Spike protein with K D <6 μM, and it is found that these compounds can be used as effective coronavirus invasion inhibitors.
本发明包括药物组合物,该组合物含有式Ⅰ所示化合物或其药学上可接受的盐,或其对应异构体、非对应异构体或外消旋体作为活性成分,以及药学上可接受的赋型剂。所述药学上可接受的赋形剂是指任何可用于药学领域的稀释剂、辅助剂或载体。本发明化合物可以与其他活性成分组合使用,只要它们不产生其他的不利作用。The present invention includes a pharmaceutical composition comprising a compound represented by formula I or a pharmaceutically acceptable salt thereof, or an enantiomer, diastereomer or racemate thereof as an active ingredient, and a pharmaceutically acceptable Accepted excipients. The pharmaceutically acceptable excipient refers to any diluent, adjuvant or carrier that can be used in the pharmaceutical field. The compounds of the present invention may be used in combination with other active ingredients as long as they do not produce other adverse effects.
有益效果:本发明的化合物能够结合SARS-CoV-2的刺突蛋白上与ACE2受体结合的RBD区域,高效抑制SARS-CoV-2的RBD区域与ACE2相互作用,阻止病毒进入细胞,同时能够促使Spike-ACE2复合物的解离,可用于制备治疗和/或预防SARS-CoV-2新型冠状病毒感染的药物。Beneficial effects: The compound of the present invention can bind to the RBD region on the spike protein of SARS-CoV-2 that binds to the ACE2 receptor, effectively inhibits the interaction between the RBD region of SARS-CoV-2 and ACE2, prevents the virus from entering cells, and at the same time can Promoting the dissociation of the Spike-ACE2 complex can be used to prepare a drug for treating and/or preventing SARS-CoV-2 novel coronavirus infection.
图1为化合物1剂量依赖性阻断SARS-CoV-2 RBD和ACE2蛋白的相互作用及半抑制浓度测定结果;Figure 1 shows the dose-dependent blocking of the interaction between SARS-CoV-2 RBD and ACE2 protein by compound 1 and the measurement results of the half-inhibitory concentration;
图2为化合物2剂量依赖性阻断SARS-CoV-2 RBD和ACE2蛋白的相互作用及半抑制浓度测定结果;Figure 2 shows the dose-dependent blocking of the interaction between SARS-CoV-2 RBD and ACE2 protein by compound 2 and the results of the half-inhibitory concentration assay;
图3为化合物3剂量依赖性阻断SARS-CoV-2 RBD和ACE2蛋白的相互作用及半抑制浓度测定结果;Figure 3 shows the dose-dependent blocking of the interaction between SARS-CoV-2 RBD and ACE2 protein by compound 3 and the results of the half-inhibitory concentration assay;
图4为本发明化合物促进Spike与ACE2复合物解离活性测定结果,(其中:**P<0.01,***P<0.001vs.溶剂对照组);Figure 4 is the assay result of the compound of the present invention promoting the dissociation activity of Spike and ACE2 complex, (wherein: **P<0.01, ***P<0.001 vs. solvent control group);
图5为化合物1与SARS-CoV-2 RBD结合的亲和力测定结果;Figure 5 shows the results of affinity determination of compound 1 binding to SARS-CoV-2 RBD;
图6为化合物2与SARS-CoV-2 RBD结合的亲和力测定结果;Figure 6 shows the results of affinity determination of compound 2 binding to SARS-CoV-2 RBD;
图7为化合物3与SARS-CoV-2 RBD结合的亲和力测定结果;Figure 7 is the result of affinity determination of compound 3 binding to SARS-CoV-2 RBD;
图8为重组人ACE2蛋白对293T-ACE2细胞的毒性测定结果;Figure 8 shows the results of the toxicity assay of recombinant human ACE2 protein on 293T-ACE2 cells;
图9为化合物1对过表达ACE2受体的293T(293T-ACE2)细胞的毒性测定结果;Figure 9 shows the results of the toxicity assay of compound 1 on 293T (293T-ACE2) cells overexpressing the ACE2 receptor;
图10为化合物2对293T-ACE2细胞的毒性测定结果;Figure 10 shows the results of the toxicity assay of compound 2 on 293T-ACE2 cells;
图11为化合物3对293T-ACE2细胞的毒性测定结果;Figure 11 shows the results of the toxicity assay of compound 3 on 293T-ACE2 cells;
图12为化合物1、2、3抑制SARS-CoV-2新型冠状病毒假病毒入侵293T-ACE2细胞的荧光显微镜观察图;Figure 12 is a fluorescence microscope observation of compounds 1, 2, and 3 inhibiting SARS-CoV-2 novel coronavirus pseudovirus from invading 293T-ACE2 cells;
图13为重组人ACE2蛋白剂量依赖性抑制SARS-CoV-2新型冠状病毒假病毒入侵293T-ACE2细胞及半抑制浓度测定结果;Figure 13 shows the dose-dependent inhibition of the SARS-CoV-2 novel coronavirus pseudovirus from invading 293T-ACE2 cells by recombinant human ACE2 protein and the assay results of the half-inhibitory concentration;
图14为化合物1剂量依赖性抑制SARS-CoV-2新型冠状病毒假病毒入侵293T-ACE2细胞及半抑制浓度测定结果;Figure 14 shows the dose-dependent inhibition of SARS-CoV-2 novel coronavirus pseudovirus from invading 293T-ACE2 cells and the assay results of the half-inhibitory concentration;
图15为化合物2剂量依赖性抑制SARS-CoV-2新型冠状病毒假病毒入侵293T-ACE2 细胞及半抑制浓度测定结果;Figure 15 shows the dose-dependent inhibition of the SARS-CoV-2 novel coronavirus pseudovirus from invading 293T-ACE2 cells and the half-inhibitory concentration assay results of compound 2;
图16为化合物3剂量依赖性抑制SARS-CoV-2新型冠状病毒假病毒入侵293T-ACE2细胞及半抑制浓度测定结果;Figure 16 shows the dose-dependent inhibition of the SARS-CoV-2 novel coronavirus pseudovirus from invading 293T-ACE2 cells and the half-inhibitory concentration assay results of compound 3;
图17为仅加入新型冠状病毒S蛋白假病毒的模型对照组;Figure 17 is a model control group that only added the new coronavirus S protein pseudovirus;
图18为加入新型冠状病毒S蛋白假病毒与180nM ACE2重组蛋白混合物的阳性对照组;Figure 18 is a positive control group added with a mixture of novel coronavirus S protein pseudovirus and 180nM ACE2 recombinant protein;
图19为加入新型冠状病毒S蛋白假病毒与5μM化合物2混合物的实验组;Figure 19 is the experimental group added with a mixture of novel coronavirus S protein pseudovirus and 5 μM compound 2;
图20为本发明化合物2剂量依赖性抑制SARS-CoV-2新型冠状病毒假病毒入侵Calu-3细胞及半抑制浓度测定结果。Figure 20 shows the dose-dependent inhibition of SARS-CoV-2 novel coronavirus pseudovirus intrusion into Calu-3 cells and the half-inhibitory concentration determination results of compound 2 of the present invention.
实施例1Example 1
本发明化合物的制备与结构鉴定。Preparation and structure identification of the compounds of the present invention.
下面将以三个化合物为例(结构如下所示):Three compounds will be used as examples below (the structures are shown below):
实验方法:experimental method:
(一)化合物1的制备方法,如下:(1) The preparation method of compound 1 is as follows:
(1)取草麻黄的干燥草质茎,50℃烘干,粉碎后过65目筛,取筛下麻黄粉末备用;(1) get the dry herbaceous stem of Ephedra japonica, dry at 50 DEG C, pass through a 65 mesh sieve after pulverizing, get the ephedra ephedra powder under the sieve for subsequent use;
(2)向麻黄粉末中加入其重量50倍的重量百分比浓度为70%的乙醇进行超声波辅助提取,提取温度为25℃,提取时间为0.5h,超声频率为40KHz,过滤,去除溶剂,得到浸膏备用;(2) adding 50 times its weight of ethanol with a concentration of 70% by weight to the ephedra powder to carry out ultrasonic-assisted extraction, the extraction temperature was 25° C., the extraction time was 0.5h, the ultrasonic frequency was 40KHz, filtered, and the solvent was removed to obtain a dipping solution. Spare ointment;
(3)将步骤(2)中的产物溶解于蒸馏水中,超滤离心0.5h,取下层溶液,减压浓缩、回收溶剂,经过高效液相色谱分离得到化合物1。(3) Dissolving the product in step (2) in distilled water, ultrafiltration and centrifugation for 0.5h, taking the lower layer solution, concentrating under reduced pressure, recovering the solvent, and separating by high performance liquid chromatography to obtain compound 1.
其中上述步骤(3)所述高效液相分离条件包括:色谱柱规格为C18,5μm,4.6×250mmGracevisionHT;流动相为0.1%甲酸水溶液(A)和乙腈(B),梯度洗脱程序为0-10min,5%A;10–50min,5%–20%A;50–60min,20%–25%A;60–75min,25–35%A;75–76min,35%–5%A;76–85min,5%A。流速为1.0ml/min,检测波长为254nm,柱温保持在35℃,在高效液相上的保留时间约为8.4min。The high-performance liquid phase separation conditions described in the above step (3) include: the chromatographic column specification is C18, 5 μm, 4.6×250 mm GracevisionHT; the mobile phase is 0.1% formic acid aqueous solution (A) and acetonitrile (B), and the gradient elution program is 0- 10min, 5%A; 10–50min, 5%–20%A; 50–60min, 20%–25%A; 60–75min, 25–35%A; 75–76min, 35%–5%A; 76 –85min, 5%A. The flow rate was 1.0ml/min, the detection wavelength was 254nm, the column temperature was kept at 35°C, and the retention time on the high-performance liquid phase was about 8.4min.
(二)化合物2的制备方法,如下:(2) the preparation method of compound 2 is as follows:
(1)取草麻黄的干燥草质茎,50℃烘干,粉碎后过65目筛,取筛下麻黄粉末备用;(1) get the dry herbaceous stem of Ephedra japonica, dry at 50 DEG C, pass through a 65 mesh sieve after pulverizing, get the ephedra ephedra powder under the sieve for subsequent use;
(2)向麻黄粉末中加入其重量50倍的重量百分比浓度为70%的乙醇进行超声波辅助提取,提取温度为25℃,提取时间为0.5h,超声频率为40KHz,过滤,去除溶剂,得到浸膏备用;(2) adding 50 times its weight of ethanol with a concentration of 70% by weight to the ephedra powder to carry out ultrasonic-assisted extraction, the extraction temperature was 25° C., the extraction time was 0.5h, the ultrasonic frequency was 40KHz, filtered, and the solvent was removed to obtain a dipping solution. Spare ointment;
(3)将步骤(2)中的产物溶解于蒸馏水中,超滤离心0.5h,取下层溶液,减压浓缩、回收溶剂,经过高效液相色谱分离得到化合物2。(3) Dissolving the product in step (2) in distilled water, ultrafiltration and centrifugation for 0.5h, taking the lower layer solution, concentrating under reduced pressure, recovering the solvent, and separating by high performance liquid chromatography to obtain compound 2.
其中上述步骤(3)所述高效液相分离条件包括:色谱柱规格为C18,5μm,4.6×250mm Grace visionHT;流动相为0.1%甲酸水溶液(A)和乙腈(B),梯度洗脱程序为0-10min,5%A;10–50min,5%–20%A;50–60min,20%–25%A;60–75min,25–35%A;75–76min,35%–5%A;76–85min,5%A。流速为1.0ml/min,检测波长为254nm,柱温保持在35℃,在高效液相上的保留时间约为13.9min。The high-performance liquid phase separation conditions described in the above step (3) include: the chromatographic column specification is C18, 5μm, 4.6×250mm Grace visionHT; the mobile phase is 0.1% formic acid aqueous solution (A) and acetonitrile (B), and the gradient elution program is 0-10min, 5%A; 10–50min, 5%–20%A; 50–60min, 20%–25%A; 60–75min, 25–35%A; 75–76min, 35%–5%A ;76–85min, 5%A. The flow rate was 1.0ml/min, the detection wavelength was 254nm, the column temperature was kept at 35°C, and the retention time on the high-performance liquid phase was about 13.9min.
(三)化合物3的制备方法,如下:(3) the preparation method of compound 3, as follows:
(1)取草麻黄的干燥草质茎,50℃烘干,粉碎后过65目筛,取筛下麻黄粉末备用;(1) get the dry herbaceous stem of Ephedra japonica, dry at 50 DEG C, pass through a 65 mesh sieve after pulverizing, get the ephedra ephedra powder under the sieve for subsequent use;
(2)向麻黄粉末中加入其重量50倍的重量百分比浓度为70%的乙醇进行超声波辅助提取,提取温度为25℃,提取时间为0.5h,超声频率为40KHz,过滤,去除溶剂,得到浸膏备用;(2) adding 50 times its weight of ethanol with a concentration of 70% by weight to the ephedra powder to carry out ultrasonic-assisted extraction, the extraction temperature was 25° C., the extraction time was 0.5h, the ultrasonic frequency was 40KHz, filtered, and the solvent was removed to obtain a dipping solution. Spare ointment;
(3)将步骤(2)中的产物溶解于蒸馏水中,超滤离心0.5h,取下层溶液,减压浓缩、回收溶剂,经过高效液相色谱分离得到化合物3。(3) Dissolving the product in step (2) in distilled water, ultrafiltration and centrifugation for 0.5 h, taking the lower layer solution, concentrating under reduced pressure, recovering the solvent, and separating by high performance liquid chromatography to obtain compound 3.
其中上述步骤(3)所述高效液相分离条件包括:色谱柱规格为C18,5μm,4.6×250mm Grace visionHT;流动相为0.1%甲酸水溶液(A)和乙腈(B),梯度洗脱程序为0-10min,5%A;10–50min,5%–20%A;50–60min,20%–25%A;60–75min,25–35%A;75–76min,35%–5%A;76–85min,5%A。流速为1.0ml/min,检测波长为254nm,柱温保持在35℃,在高效液相上的保留时间约为24.7min。The high-performance liquid phase separation conditions described in the above step (3) include: the chromatographic column specification is C18, 5μm, 4.6×250mm Grace visionHT; the mobile phase is 0.1% formic acid aqueous solution (A) and acetonitrile (B), and the gradient elution program is 0-10min, 5%A; 10–50min, 5%–20%A; 50–60min, 20%–25%A; 60–75min, 25–35%A; 75–76min, 35%–5%A ;76–85min, 5%A. The flow rate was 1.0ml/min, the detection wavelength was 254nm, the column temperature was kept at 35°C, and the retention time on the high-performance liquid phase was about 24.7min.
实验结果:化合物1,HR-ESI-MS(positive)给出m/z206.0437[M+H]+,确定分子式为C
10H
7NO
4,化合物的1H-NMR(500MHz,inDMSO)谱显示7.86(d,J=9.0Hz,1H),7.39(d,J=2.5Hz,1H),7.24(dd,J=8.9,2.5Hz,1H),6.61(s,1H)。通过与标准物质对比,确定化合物1为6-hydroxykynurenic acid。化合物2,HR-ESI-MS(positive)给出m/z190.0487[M+H]+,确定分子式为C
10H
7NO
3,化合物的1H-NMR(500MHz,in DMSO)谱显示8.09(d,J=7.5Hz,1H),7.96(d,J=8.4Hz,1H),7.70(t,J=8.2Hz,1H),7.37(t,J=7.5Hz,1H),6.65(s,1H)。通过与标准物质对比,确定化合物2为kynurenic acid。化合物3,HR-ESI-MS(positive)给出m/z 220.0592[M+H]+,确定分子式为C
11H
9NO
4,化合物的1H-NMR(500MHz,in DMSO)谱显示7.92(d,J=9.1Hz,1H),7.47(d,J=2.9Hz,1H),7.34(dd,J=9.3,2.8Hz,1H),6.60(s,1H),3.85(s,3H)。通过与标准物质对比,确定化合物3为6-methoxykynurenic acid。
Experimental results: Compound 1, HR-ESI-MS (positive) gives m/z 206.0437[M+H]+, the molecular formula is determined to be C 10 H 7 NO 4 , the 1H-NMR (500MHz, inDMSO) spectrum of the compound shows 7.86 (d, J=9.0Hz, 1H), 7.39 (d, J=2.5Hz, 1H), 7.24 (dd, J=8.9, 2.5Hz, 1H), 6.61 (s, 1H). By comparing with the standard material, it was determined that compound 1 was 6-hydroxykynurenic acid. Compound 2, HR-ESI-MS (positive) gave m/z 190.0487 [M+H]+, the molecular formula was determined to be C 10 H 7 NO 3 , the 1H-NMR (500MHz, in DMSO) spectrum of the compound showed 8.09 ( d, J=7.5Hz, 1H), 7.96(d, J=8.4Hz, 1H), 7.70(t, J=8.2Hz, 1H), 7.37(t, J=7.5Hz, 1H), 6.65(s, 1H). Compound 2 was determined to be kynurenic acid by comparison with the standard material. Compound 3, m/z 220.0592 [M+H]+ by HR-ESI-MS (positive), identified as C 11 H 9 NO 4 , 1H-NMR (500 MHz, in DMSO) spectrum of the compound showed 7.92 (d , J=9.1Hz, 1H), 7.47 (d, J=2.9Hz, 1H), 7.34 (dd, J=9.3, 2.8Hz, 1H), 6.60 (s, 1H), 3.85 (s, 3H). By comparing with the standard substance, the compound 3 was determined to be 6-methoxykynurenic acid.
实施例2Example 2
本发明所述化合物抑制SARS-CoV-2新型冠状病毒Spike蛋白RBD区域与ACE2受体相互作用。The compound of the present invention inhibits the interaction between the RBD region of the SARS-CoV-2 novel coronavirus Spike protein and the ACE2 receptor.
实验方法:竞争性结合抑制实验。向96孔微孔板中加入100μl 0.5μg/ml溶解于包被液(50mM碳酸盐缓冲液,pH 9.6)的SARS-Cov-2 RBD重组蛋白,于4℃放置过夜。第二天,用300μl洗涤液(含0.05%Tween 20的磷酸盐缓冲液,pH 7.4)漂洗三次,去除未包被的蛋白。随后加入300μl封闭液(含2%牛血清白蛋白的洗涤液,pH 7.4)并在37℃条件下于封闭1h。漂洗三次后,将50μl不同浓度待测化合物与50μl的0.12μg/ml生物素化的ACE2混合后加入96孔板,并在37℃孵育1h。漂洗三次后,将100μl的链霉亲和素标记的辣根过氧化 物酶加入到每个孔中,并在37℃孵育1h。漂洗三次后,将200μl显色液(含0.1mg/ml TMB以及0.004%过氧化氢的磷酸氢钠-柠檬酸缓冲液,pH5.5)添加到每个孔中,并在37℃避光孵育20分钟。加入50μl反应终止液(1M硫酸)后,使用酶标仪在450nm处读取吸光度。使用GraphPad Prism 6.0软件分析原始数据,并计算相对抑制率。测定值是用三次独立实验的平均值±平均值的标准误差表示。Experimental method: Competitive binding inhibition assay. 100 μl of 0.5 μg/ml SARS-Cov-2 RBD recombinant protein dissolved in coating solution (50 mM carbonate buffer, pH 9.6) was added to a 96-well microplate, and placed at 4°C overnight. The next day, uncoated proteins were removed by rinsing three times with 300 μl of washing solution (phosphate buffer containing 0.05% Tween 20, pH 7.4). 300 μl of blocking solution (2% bovine serum albumin in wash, pH 7.4) was then added and blocked for 1 h at 37°C. After rinsing three times, 50 μl of compounds to be tested at different concentrations were mixed with 50 μl of 0.12 μg/ml biotinylated ACE2 and added to a 96-well plate, and incubated at 37° C. for 1 h. After three washes, 100 [mu]l of streptavidin-labeled horseradish peroxidase was added to each well and incubated at 37[deg.]C for 1 h. After three washes, 200 μl of chromogenic solution (sodium hydrogen phosphate-citrate buffer containing 0.1 mg/ml TMB and 0.004% hydrogen peroxide, pH 5.5) was added to each well and incubated at 37°C in the dark 20 minutes. After adding 50 μl of reaction stop solution (1M sulfuric acid), the absorbance was read at 450 nm using a microplate reader. Raw data were analyzed using GraphPad Prism 6.0 software, and relative inhibition rates were calculated. Measured values are expressed as the mean ± standard error of the mean of three independent experiments.
实验结果:如图1-3所示,化合物1-3均剂量依赖性阻断SARS-CoV-2 RBD和ACE2重组蛋白的相互作用,半抑制浓度分别0.96μM,0.10μM和0.16μM。本发明所述化合物能够高效抑制SARS-CoV-2新型冠状病毒Spike蛋白与ACE2受体相互作用。Experimental results: As shown in Figures 1-3, compounds 1-3 blocked the interaction between SARS-CoV-2 RBD and ACE2 recombinant protein in a dose-dependent manner, with half-inhibitory concentrations of 0.96 μM, 0.10 μM and 0.16 μM, respectively. The compound of the invention can effectively inhibit the interaction between the SARS-CoV-2 novel coronavirus Spike protein and the ACE2 receptor.
实施例3Example 3
本发明所述化合物促进SARS-CoV-2新型冠状病毒Spike蛋白RBD区域与ACE2受体复合物的解离。The compound of the present invention promotes the dissociation of the SARS-CoV-2 novel coronavirus Spike protein RBD region and the ACE2 receptor complex.
实验方法:竞争性结合抑制实验。向96孔微孔板中加入100μl 0.5μg/ml溶解于包被液(50mM碳酸盐缓冲液,pH 9.6)的SARS-CoV-2 RBD重组蛋白,于4℃放置过夜。第二天,用300μl洗涤液(含0.05%Tween 20的磷酸盐缓冲液,pH 7.4)漂洗三次,去除未包被的蛋白。随后加入300μl封闭液(含2%牛血清白蛋白的洗涤液,pH 7.4)并在37℃条件下于封闭1h。漂洗三次后,将50μl的0.12μg/ml生物素化的ACE2加入96孔板,并在37℃孵育1h。漂洗三次后,将50μl浓度为25μM的待测化合物加入96孔板,并在37℃孵育1h。漂洗三次后,将100μl的链霉亲和素标记的辣根过氧化物酶加入到每个孔中,并在37℃孵育1h。漂洗三次后,将200μl显色液(含0.1mg/ml TMB以及0.004%过氧化氢的磷酸氢钠-柠檬酸缓冲液,pH5.5)添加到每个孔中,并在37℃避光孵育20分钟。加入50μl反应终止液(1M硫酸)后,使用酶标仪在450nm处读取吸光度。使用GraphPad Prism 6.0软件分析原始数据。测定值是用三次独立实验的平均值±平均值的标准误差表示。组间分析采用单因素方差分析,p<0.05被认为具有统计学意义。Experimental method: Competitive binding inhibition assay. 100 μl of 0.5 μg/ml SARS-CoV-2 RBD recombinant protein dissolved in coating solution (50 mM carbonate buffer, pH 9.6) was added to a 96-well microplate and placed at 4°C overnight. The next day, uncoated proteins were removed by rinsing three times with 300 μl of washing solution (phosphate buffer containing 0.05% Tween 20, pH 7.4). 300 μl of blocking solution (2% bovine serum albumin in wash, pH 7.4) was then added and blocked for 1 h at 37°C. After rinsing three times, 50 μl of 0.12 μg/ml biotinylated ACE2 was added to the 96-well plate and incubated for 1 h at 37°C. After rinsing three times, 50 μl of the test compound at a concentration of 25 μM was added to the 96-well plate and incubated at 37° C. for 1 h. After three washes, 100 μl of streptavidin-labeled horseradish peroxidase was added to each well and incubated for 1 h at 37°C. After three washes, 200 μl of chromogenic solution (sodium hydrogen phosphate-citrate buffer containing 0.1 mg/ml TMB and 0.004% hydrogen peroxide, pH 5.5) was added to each well and incubated at 37°C in the dark 20 minutes. After adding 50 μl of reaction stop solution (1M sulfuric acid), the absorbance was read at 450 nm using a microplate reader. Raw data were analyzed using GraphPad Prism 6.0 software. Measured values are expressed as the mean ± standard error of the mean of three independent experiments. Between-group analysis was performed using one-way ANOVA, and p<0.05 was considered statistically significant.
实验结果:如图4所示,分别加入化合物1-3之后,与溶剂对照组相比与SARS-CoV-2RBD结合的ACE2的量明显减少。本发明所述化合物促进SARS-CoV-2新型冠状病毒Spike蛋白RBD区域与ACE2受体复合物的解离。Experimental results: As shown in Figure 4, after adding compounds 1-3 respectively, the amount of ACE2 bound to SARS-CoV-2 RBD was significantly reduced compared with the solvent control group. The compound of the present invention promotes the dissociation of the SARS-CoV-2 novel coronavirus Spike protein RBD region and the ACE2 receptor complex.
实施例4Example 4
本发明所述化合物与SARS-CoV-2新型冠状病毒Spike蛋白RBD区域相互作用。The compound of the present invention interacts with the RBD region of the SARS-CoV-2 novel coronavirus Spike protein.
实验方法:表面等离子共振(SPR)实验。Experimental method: Surface Plasmon Resonance (SPR) experiment.
SPR传感技术,利用表面等离子体共振的原理,当光耦合到传感芯片时,由于发生表面等离子体共振,一部分光能量被吸收,使得从传感芯片反射的光形成SPR共振信号。SPR共振信号对于传感芯片表面样品物质折射率的变化非常敏感,因此就可以通过分析SPR共振图而获得样品的物质信息。在检测蛋白-蛋白相互作用等生物传感领域有极其重要的应用。SPR sensing technology uses the principle of surface plasmon resonance. When light is coupled to the sensor chip, due to the occurrence of surface plasmon resonance, part of the light energy is absorbed, so that the light reflected from the sensor chip forms an SPR resonance signal. The SPR resonance signal is very sensitive to the change of the refractive index of the sample material on the sensor chip surface, so the material information of the sample can be obtained by analyzing the SPR resonance image. It has extremely important applications in the field of biosensing such as the detection of protein-protein interactions.
该实验使用BIAcore T200仪器在25℃下进行测试,流动相为磷酸盐缓冲液(pH 7.4)。用10mmol/L醋酸钠缓冲液(pH值分别为5.5、5.0、4.5及4.0)稀释SARS-CoV-2 RBD重组蛋白至终浓度为20μg/ml,采用手动进样法进行pH值筛选,选取偶联值最大的pH值进行后续实验。采用氨基偶联法将SARS-CoV-2 RBD重组蛋白偶联到CM5芯片上,以磷酸盐缓冲液为工作缓冲液,用上述筛选所得最佳pH值的10mmol/L醋酸钠缓冲液稀释SARS-CoV-2 RBD 重组蛋白至终浓度为20μg/ml。芯片表面用0.2mol/L EDC和50mmol/L NHS以1:1比例配制的混合液活化,以10μl/min的流速进样,连续进样7min后注射SARS-CoV-2 RBD重组蛋白溶液,然后以1mol/L乙醇胺盐酸(pH 8.5)封闭液进样7min,封闭活化的芯片表面,并将封闭的空白通道用作测定的阴性对照。实验选取最高浓度为25μM的本发明所述化合物作为配体,将配体以两倍连续稀释液注入固定了SARS-CoV-2 RBD重组蛋白生物传感器芯片上。1:1结合模型用于评估结合动力学。用动力学模型,通过BIAcore T200分析软件计算K
D值。
The experiments were performed at 25°C using a BIAcore T200 instrument with a mobile phase of phosphate buffered saline (pH 7.4). The SARS-CoV-2 RBD recombinant protein was diluted with 10 mmol/L sodium acetate buffer (pH values were 5.5, 5.0, 4.5 and 4.0) to a final concentration of 20 μg/ml, and the pH value was screened by manual injection method. Connect the pH value with the maximum value for subsequent experiments. The SARS-CoV-2 RBD recombinant protein was coupled to the CM5 chip by amino coupling method. Using phosphate buffer as the working buffer, SARS-CoV-2 was diluted with 10 mmol/L sodium acetate buffer with the best pH value obtained from the above screening. CoV-2 RBD recombinant protein to a final concentration of 20 μg/ml. The chip surface was activated with a mixture of 0.2 mol/L EDC and 50 mmol/L NHS in a ratio of 1:1, injected at a flow rate of 10 μl/min, and injected with SARS-CoV-2 RBD recombinant protein solution after continuous injection for 7 min. The sample was injected with 1 mol/L ethanolamine hydrochloric acid (pH 8.5) blocking solution for 7 min to block the activated chip surface, and the blocked blank channel was used as a negative control for the determination. In the experiment, the compound of the present invention with the highest concentration of 25 μM was selected as the ligand, and the ligand was injected into the immobilized SARS-CoV-2 RBD recombinant protein biosensor chip in two-fold serial dilution. A 1:1 binding model was used to assess binding kinetics. KD values were calculated by BIAcore T200 analysis software using the kinetic model.
实验结果:如图5、6、7所示,化合物1与SARS-CoV-2 RBD重组蛋白的亲和力0.60μM,化合物2与SARS-CoV-2 RBD重组蛋白的亲和力5.28μM,化合物3与SARS-CoV-2 RBD重组蛋白的亲和力5.37μM。动力学分析数据显示,与化合物2或3相比,虽然化合物1与SARS-CoV-2 RBD重组蛋白的亲和力略强,但都在一个数量级上,这一结果提示我们本发明所述化合物骨架是其发挥功能的关键结构,R
1或R2取代基的变化对化合物活性没有明显影响。式Ⅰ所示的化合物可能均具有该活性。本发明所述化合物与SARS-CoV-2新型冠状病毒Spike蛋白RBD区域相互作用。
Experimental results: As shown in Figures 5, 6, and 7, the affinity of compound 1 to SARS-CoV-2 RBD recombinant protein was 0.60 μM, the affinity of compound 2 to SARS-CoV-2 RBD recombinant protein was 5.28 μM, and the affinity of compound 3 to SARS-CoV-2 RBD recombinant protein The affinity of CoV-2 RBD recombinant protein is 5.37 μM. The kinetic analysis data showed that, compared with compound 2 or 3, although the affinity of compound 1 and SARS-CoV-2 RBD recombinant protein was slightly stronger, they were all in the same order of magnitude. This result suggests that the skeleton of the compound described in the present invention is The key structure for its function, the change of R1 or R2 substituent had no obvious effect on the activity of the compound. Compounds of formula I may all possess this activity. The compound of the present invention interacts with the RBD region of the SARS-CoV-2 novel coronavirus Spike protein.
实施例5Example 5
本发明所述化合物抑制SARS-CoV-2新型冠状病毒S蛋白假病毒入侵过表达ACE2受体的293T细胞(293T-ACE2)。The compound of the present invention inhibits the invasion of 293T cells (293T-ACE2) overexpressing the ACE2 receptor by the pseudovirus of the S protein of the SARS-CoV-2 novel coronavirus.
实验方法:新型冠状病毒S蛋白假病毒入侵细胞实验。SARS-CoV-2新型冠状病毒S蛋白假病毒含有绿色荧光蛋白报告基因,当病毒入侵细胞后可在荧光显微镜下观察到绿色荧光蛋白表达,反应了病毒入侵细胞的数量。新型冠状病毒S蛋白与ACE2受体的结合是病毒入侵细胞的关键步骤,因此选择过表达ACE2受体的293T细胞(293T-ACE2)模拟病毒入侵。首先采用MTT法,检测各个化合物对细胞的活力的影响,是否具有细胞毒性。将293T-ACE2细胞均匀铺至孔板中,细胞培养使用含10%胎牛血清的DMEM培养基,在37℃、5%CO2条件下培养24小时后,将各种化合物添加到每个孔中,再孵育24小时。然后将293T-ACE2细胞置于100μl含500μg/ml MTT的10%FBS-DMEM中。在37℃下孵育4小时后,向每个孔中加入150μl二甲基亚砜以溶解结晶。使用酶标仪在570nm处测量吸光度值。随后进行病毒入侵实验,将293T-ACE2细胞均匀铺至孔板中,细胞培养使用含10%胎牛血清的DMEM培养基,在37℃、5%CO
2条件下培养,待细胞生长到30%开始进行实验。将相同浓度的SARS-CoV-2新型冠状病毒S蛋白假病毒与不同浓度待测化合物混合后,在37℃孵育1h,随后加入到每个孔中。孵育6h后更换为新鲜培养基。正常培养48h之后,用荧光显微镜(20×)拍照观察不同细胞中绿色荧光蛋白的表达情况,并用荧光酶标仪在激发波长为488nm发射波长为520nm定量检测绿色荧光蛋白的表达量,分析病毒入侵293T-ACE2细胞的数量。使用GraphPad Prism 6.0软件分析原始数据,并计算相对抑制率。测定值是用三次独立实验的平均值±平均值的标准误差表示。
Experimental method: The new coronavirus S protein pseudovirus invades cells. The SARS-CoV-2 novel coronavirus S protein pseudovirus contains a green fluorescent protein reporter gene. When the virus invades cells, the expression of green fluorescent protein can be observed under a fluorescence microscope, which reflects the number of cells invaded by the virus. The binding of the new coronavirus S protein to the ACE2 receptor is a key step for the virus to invade cells, so 293T cells (293T-ACE2) overexpressing the ACE2 receptor were selected to simulate virus invasion. First, the MTT method was used to detect the effect of each compound on cell viability and whether it had cytotoxicity. 293T-ACE2 cells were evenly plated into well plates, and DMEM medium containing 10% fetal bovine serum was used for cell culture. After culturing for 24 hours at 37°C and 5% CO2, various compounds were added to each well. , and incubated for another 24 hours. 293T-ACE2 cells were then placed in 100 μl of 10% FBS-DMEM containing 500 μg/ml MTT. After 4 hours of incubation at 37°C, 150 μl of dimethyl sulfoxide was added to each well to dissolve the crystals. Absorbance values were measured at 570 nm using a microplate reader. Then the virus invasion experiment was carried out, 293T-ACE2 cells were evenly spread into the well plate, and the cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 . Start experimenting. The same concentration of SARS-CoV-2 novel coronavirus S protein pseudovirus was mixed with different concentrations of the test compounds, incubated at 37°C for 1 h, and then added to each well. After 6 h of incubation, the medium was replaced with fresh medium. After 48 hours of normal culture, take pictures with a fluorescence microscope (20×) to observe the expression of green fluorescent protein in different cells, and use a fluorescence microplate reader to quantitatively detect the expression of green fluorescent protein at an excitation wavelength of 488 nm and an emission wavelength of 520 nm to analyze the virus invasion. Number of 293T-ACE2 cells. Raw data were analyzed using GraphPad Prism 6.0 software, and relative inhibition rates were calculated. Measured values are expressed as the mean ± standard error of the mean of three independent experiments.
实验结果:化合物1-3均可以阻止新型冠状病毒S蛋白假病毒进入293T-ACE2细胞。其中,如图8、9、10、11所示,化合物1-3以及阳性对照hACE2重组蛋白均在一定浓度范围内对293T-ACE2细胞活力没有影响,表明该类化合物安全性良好。如图12所示,可以在荧光显微镜下明显观察到与不表达ACE2受体的293T细胞相比,模型组293T-ACE2细胞表达大量绿色荧光蛋白,表明ACE2是帮助新型冠状病毒S蛋白假病毒入侵细胞的关键受体。同时可以在荧光显微镜下观察到,与模型组相比,分别加入阳性对照ACE2重组蛋白以及化合物1-3之后, 绿色荧光蛋白表达均明显减少,说明其均可以抑制病毒入侵细胞,起到细胞保护作用。图13、14、15、16显示ACE2重组蛋白以及化合物1-3均可以剂量依赖性抑制阻止新型冠状病毒S蛋白假病毒进入293T-ACE2细胞,半抑制浓度分别为69.62nM,1.09μM,0.44μM和0.75μM。这些结果均说明本发明所述化合物抑制SARS-CoV-2新型冠状病毒S蛋白假病毒入侵过表达ACE2受体的293T细胞。Experimental results: Compounds 1-3 can prevent the entry of the new coronavirus S protein pseudovirus into 293T-ACE2 cells. Among them, as shown in Figures 8, 9, 10, and 11, compounds 1-3 and the positive control hACE2 recombinant protein had no effect on the viability of 293T-ACE2 cells within a certain concentration range, indicating that these compounds are safe. As shown in Figure 12, it can be clearly observed under the fluorescence microscope that compared with the 293T cells that do not express the ACE2 receptor, the 293T-ACE2 cells in the model group express a large amount of green fluorescent protein, indicating that ACE2 helps the new coronavirus S protein pseudovirus to invade key receptors in cells. At the same time, it can be observed under a fluorescence microscope that, compared with the model group, after adding the positive control ACE2 recombinant protein and compounds 1-3, the expression of green fluorescent protein was significantly reduced, indicating that both can inhibit virus invasion into cells and play a role in cell protection. effect. Figures 13, 14, 15, and 16 show that ACE2 recombinant protein and compounds 1-3 can dose-dependently inhibit the entry of the new coronavirus S protein pseudovirus into 293T-ACE2 cells, and the half-inhibitory concentrations are 69.62nM, 1.09μM, and 0.44μM, respectively. and 0.75 μM. These results all indicate that the compounds of the present invention inhibit the invasion of 293T cells overexpressing the ACE2 receptor by the SARS-CoV-2 novel coronavirus S protein pseudovirus.
实施例6Example 6
本发明所述化合物抑制SARS-CoV-2新型冠状病毒S蛋白假病毒入侵人肺腺上皮细胞(Calu-3)。The compound of the present invention inhibits the invasion of SARS-CoV-2 novel coronavirus S protein pseudovirus into human lung glandular epithelial cells (Calu-3).
实验方法:新型冠状病毒S蛋白假病毒入侵细胞实验。SARS-CoV-2新型冠状病毒S蛋白假病毒含有绿色荧光蛋白报告基因,当病毒入侵细胞后可在荧光显微镜下观察到绿色荧光蛋白表达,反应了病毒入侵细胞的数量。将人肺腺上皮细胞(Calu-3)均匀铺至孔板中,细胞培养使用含10%胎牛血清的MEM培养基,在37℃、5%CO
2条件下培养,待细胞生长到50%开始进行实验。将相同浓度的SARS-CoV-2新型冠状病毒S蛋白假病毒与不同浓度待测化合物混合后,在37℃孵育1h,随后加入到每个孔中。正常培养24h之后,用荧光显微镜(20×)拍照观察不同细胞中绿色荧光蛋白的表达情况,并用荧光酶标仪在激发波长为488nm发射波长为520nm定量检测绿色荧光蛋白的表达量,分析病毒入侵Calu-3细胞的数量。使用GraphPad Prism 6.0软件分析原始数据,并计算相对抑制率。测定值是用三次独立实验的平均值±平均值的标准误差表示。
Experimental method: The new coronavirus S protein pseudovirus invades cells. The SARS-CoV-2 novel coronavirus S protein pseudovirus contains a green fluorescent protein reporter gene. When the virus invades cells, the expression of green fluorescent protein can be observed under a fluorescence microscope, which reflects the number of cells invaded by the virus. Human lung gland epithelial cells (Calu-3) were evenly plated into the well plate, and the cells were cultured in MEM medium containing 10% fetal bovine serum at 37°C and 5% CO 2 , and the cells were grown to 50%. Start experimenting. The same concentration of SARS-CoV-2 novel coronavirus S protein pseudovirus was mixed with different concentrations of the test compounds, incubated at 37°C for 1 h, and then added to each well. After 24 hours of normal culture, take pictures with a fluorescence microscope (20×) to observe the expression of green fluorescent protein in different cells, and use a fluorescence microplate reader to quantitatively detect the expression of green fluorescent protein at an excitation wavelength of 488 nm and an emission wavelength of 520 nm to analyze the virus invasion. Number of Calu-3 cells. Raw data were analyzed using GraphPad Prism 6.0 software, and relative inhibition rates were calculated. Measured values are expressed as the mean ± standard error of the mean of three independent experiments.
实验结果:如图17、18、19为化合物2抑制SARS-CoV-2新型冠状病毒假病毒入侵人肺腺上皮细胞(Calu-3)的荧光显微镜观察图,如图所示,化合物2可以阻止新型冠状病毒S蛋白假病毒进入Calu-3细胞。其中,图17为仅加入新型冠状病毒S蛋白假病毒的模型对照组。图18为加入新型冠状病毒S蛋白假病毒与180nM ACE2重组蛋白混合物的阳性对照组。图19为加入新型冠状病毒S蛋白假病毒与5μM化合物2混合物的实验组。可以在荧光显微镜下观察到,与模型对照组相比,加入阳性对照ACE2重组蛋白以及化合物2之后,绿色荧光蛋白表达明显减弱,二者均可以抑制病毒入侵细胞,起到保护作用。图20显示化合物2可以剂量依赖性抑制阻止新型冠状病毒S蛋白假病毒进入Calu-3细胞,半抑制浓度为322.6nM。这些结果均说明本发明所述化合物抑制SARS-CoV-2新型冠状病毒S蛋白假病毒入侵人肺腺上皮细胞。Experimental results: Figures 17, 18, and 19 are fluorescence microscope observations of compound 2 inhibiting SARS-CoV-2 novel coronavirus pseudovirus from invading human lung glandular epithelial cells (Calu-3). As shown in the figure, compound 2 can prevent The novel coronavirus S protein pseudovirus enters Calu-3 cells. Among them, Figure 17 is a model control group with only the new coronavirus S protein pseudovirus added. Figure 18 is a positive control group added with a mixture of novel coronavirus S protein pseudovirus and 180nM ACE2 recombinant protein. Figure 19 shows the experimental group added with a mixture of novel coronavirus S protein pseudovirus and 5 μM compound 2. It can be observed under a fluorescence microscope that compared with the model control group, the expression of green fluorescent protein was significantly weakened after the addition of the positive control ACE2 recombinant protein and compound 2, both of which could inhibit the virus from invading cells and play a protective role. Figure 20 shows that compound 2 can dose-dependently inhibit the entry of the novel coronavirus S protein pseudovirus into Calu-3 cells, with a half-inhibitory concentration of 322.6 nM. These results all indicate that the compounds of the present invention inhibit the invasion of human lung gland epithelial cells by the SARS-CoV-2 novel coronavirus S protein pseudovirus.
Claims (7)
- 一种能够抑制冠状病毒Spike蛋白与ACE2相互作用的化合物在制备治疗和/或预防SARS-CoV-2新型冠状病毒感染的药物中的用途,化合物为如下几种:Use of a compound capable of inhibiting the interaction between coronavirus Spike protein and ACE2 in the preparation of a medicine for treating and/or preventing SARS-CoV-2 novel coronavirus infection, the compounds are as follows:
- 根据权利要求1所述的用途,其特征在于:化合物1、化合物2、化合物3的半数抑制浓度分别为0.96μM、0.10μM和0.16μM。The use according to claim 1, wherein the median inhibitory concentrations of compound 1, compound 2 and compound 3 are 0.96 μM, 0.10 μM and 0.16 μM, respectively.
- 根据权利要求1所述的用途,其特征在于:化合物能特异性的结合在Spike蛋白的RBD区域。The use according to claim 1, wherein the compound can specifically bind to the RBD region of the Spike protein.
- 根据权利要求1所述的用途,其特征在于:化合物可以抑制冠状病毒Spike蛋白与ACE2蛋白的结合或促使Spike-ACE2复合物的解离。The use according to claim 1, wherein the compound can inhibit the binding of coronavirus Spike protein and ACE2 protein or promote the dissociation of Spike-ACE2 complex.
- 根据权利要求1所述的用途,所述药物剂型为口服给药剂型或非口服给药剂型。The use according to claim 1, wherein the pharmaceutical dosage form is an oral dosage form or a parenteral dosage form.
- 根据权利要求5所述的用途,所述的口服给药剂型为片剂、散剂、颗粒剂、胶囊剂、乳剂或糖浆剂。The use according to claim 5, the oral dosage form is tablet, powder, granule, capsule, emulsion or syrup.
- 根据权利要求5所述的用途,所述的非口服给药剂型是注射剂。The use according to claim 5, wherein the parenteral dosage form is an injection.
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Title |
---|
ALEXPANDI RAJAIAH, DE MESQUITA JOELMA FREIRE, PANDIAN SHUNMUGIAH KARUTHA, RAVI ARUMUGAM VEERA: "Quinolines-Based SARS-CoV-2 3CLpro and RdRp Inhibitors and Spike-RBD-ACE2 Inhibitor for Drug-Repurposing Against COVID-19: An in silico Analysis", FRONTIERS IN MICROBIOLOGY, vol. 11, 23 July 2020 (2020-07-23), pages 1 - 15, XP055946543, DOI: 10.3389/fmicb.2020.01796 * |
GAUDÊNCIO SUSANA P., PEREIRA FLORBELA: "A Computer-Aided Drug Design Approach to Predict Marine Drug-Like Leads for SARS-CoV-2 Main Protease Inhibition", MARINE DRUGS, vol. 18, no. 12, 1 January 2020 (2020-01-01), pages 1 - 17, XP055946545, DOI: 10.3390/md18120633 * |
GENTILE DAVIDE, FUOCHI VIRGINIA, RESCIFINA ANTONIO, FURNERI PIO MARIA: "New Anti SARS-Cov-2 Targets for Quinoline Derivatives Chloroquine and Hydroxychloroquine", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 21, no. 16, 1 January 2020 (2020-01-01), pages 1 - 16, XP055946547, DOI: 10.3390/ijms21165856 * |
MEI JIE, ZHOU YATONG, YANG XINPING, ZHANG FAN, LIU XIUFENG, YU BOYANG: "Active components in Ephedra sinica stapf disrupt the interaction between ACE2 and SARS-CoV-2 RBD: Potent COVID-19 therapeutic agents", JOURNAL OF ETHNOPHARMACOLOGY, vol. 278, 1 October 2021 (2021-10-01), IE , pages 1 - 10, XP055917015, ISSN: 0378-8741, DOI: 10.1016/j.jep.2021.114303 * |
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