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Mutations in spike protein and allele variations in ACE2 impact targeted therapy strategies against SARS-CoV-2

Chuan-Jun Shu Xuan Huang Hui-Hao Tang Ding-Ding Mo Jian-Wei Zhou Cheng Deng

Chuan-Jun Shu, Xuan Huang, Hui-Hao Tang, Ding-Ding Mo, Jian-Wei Zhou, Cheng Deng. Mutations in spike protein and allele variations in ACE2 impact targeted therapy strategies against SARS-CoV-2. Zoological Research, 2021, 42(2): 170-181. doi: 10.24272/j.issn.2095-8137.2020.301
Citation: Chuan-Jun Shu, Xuan Huang, Hui-Hao Tang, Ding-Ding Mo, Jian-Wei Zhou, Cheng Deng. Mutations in spike protein and allele variations in ACE2 impact targeted therapy strategies against SARS-CoV-2. Zoological Research, 2021, 42(2): 170-181. doi: 10.24272/j.issn.2095-8137.2020.301

刺突蛋白的突变和血管紧张素转化酶2的等位基因变异对SARS-CoV-2靶向治疗策略的影响

doi: 10.24272/j.issn.2095-8137.2020.301

Mutations in spike protein and allele variations in ACE2 impact targeted therapy strategies against SARS-CoV-2

Funds: This work was supported by the National Key Research and Development Program of China (2018YFD0900602), National Natural Science Foundation of China (31970388, 31701234), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Natural Science Foundation of the Jiangsu Higher Education Institutions (17KJB180006), Natural Science Foundation from Jiangsu Province (BK20160043, BK20151546, 15KJA180004 and BK20171035), and Jiangsu Distinguished Professor Funding
More Information
  • 摘要: 新型冠状病毒肺炎(COVID-19)是由严重急性呼吸系统综合征冠状病毒2 (SARS-CoV-2)引起的一种大流行疾病,目前正在全球范围内迅速蔓延,其传播速度快,死亡人数高。迄今为止,尚无针对COVID-19的有效治疗或疫苗。血管紧张素转化酶2 (ACE2)和刺突蛋白(spike)在COVID-19治疗中的作用是当前的研究重点。在本研究中,我们探索了ACE2和spike作为靶点开发抗SARS-CoV-2病毒药物的潜力。我们分析了临床数据、测序数据和受体结合能力。其中,临床资料显示,COVID-19合并肾素-血管紧张素系统异常的患者早期症状多,预后差。然而,ACE2表达水平与COVID-19进展之间的关系尚不清楚。因此,ACE2可能不是一个很好的靶标适用于治疗不同人群的COVID-19。根据序列分析,与ACE2相互作用的spike-S1受体结合区域有许多氨基酸突变。通过受体-配体对接和ELISA试验,我们鉴定了两个spike-S1点突变体(V354F和V470A)。这些变异增强了spike蛋白与ACE2受体的结合,可能与突变体传染性增加有关。更重要的是,随着SARS-CoV-2的流行,感染V354F或V470A突变体的患者数量正在增加。这些结果表明,ACE2和spike-S1可能不适用设计广泛用于治疗不同人群COVID-19的药物理想靶点。
    #Authors contributed equally to this work
  • Figure  1.  Clinical data on COVID-19 patients who died

    A: Comorbidities in COVID-19 deaths. B: Age distribution in three groups, i.e., G1 (COVID-19 patients with comorbidities associated with abnormal RAS, 75.00±2.57 (mean±SEM (standard error of the mean))), G2 (COVID-19 patients with comorbidities but normal RAS, 70.50±4.44), and G3 (COVID-19 patients without comorbidities, 65.29±3.18). For age distribution, P-values between G1 and G2, G1 and G3, and G2 and G3 were 0.79, 0.80, and 0.70, respectively. C: Days of symptoms until death (time from early symptoms to death in G1 (15.47±1.71), G2 (16.50±2.06), and G3 (15.43±2.20) groups). For days of symptoms until death, P-values between G1 and G2, G1 and G3, and G2 and G3 were 0.36, 0.67, and 0.28, respectively. D: Sex ratio in three groups (G1-male (0.68), G2-male (0.75), G3-male (0.79); G1-female (0.32), G2-female (0.25), G3-female (0.21)). E: Early symptoms for patients in three groups (fever (G1: 0.74, G2: 1, G3: 0.79); cough (G1: 0.53, G2: 0.75, G3: 0.57); dyspnea (G1: 0.53, G2: 0.50, G3: 0.29); fatigue (G1: 0.26, G2: 0.50, G3: 0.14); diarrhea (G1: 0.05); chills (G1: 0.05); and headache (G1: 0.11).

    Figure  2.  Variants in binding regions of ACE2 and spike-S1

    A: Binding sites in ACE2 and spike-S1. Green and cyan schematic represents 3D structure of ACE2 and spike-S1, respectively. Yellow letters and blue spheres show spatial positions for ACE2-binding sites in spike-S1. Black letters and red spheres show spatial positions for spike-binding sites in ACE2. B: Spike-binding domain in ACE2. C: Nineteen amino acid substitutions in ACE2. D: Allele frequency for K26R in ACE2 for different populations. AFR, AMR, EAS, EUR, SAS, NFE, ALSPAC, TWINSUK, and OTH represent African/African American, Admixed American, East Asian, Europeans, South Asian, Non-Finnish European, Avon Longitudinal Study of Parents and Children, UK Adult Twin registry, and Other (population not assigned), respectively. E: ACE2-binding domain in spike-S1. F: Amino acid substitutions in ACE2-binding domain for spike-S1 and their corresponding frequencies.

    Figure  4.  Structural pharmacological analysis among three spike-S1 protein mutants

    A: RMD domain in spike-S1. B: Distribution of three spike mutations, i.e., N341D, D351Y, and V354F, in 3D structure. C: Physical and chemical parameters for spike-S1 and its mutants. D: Energy and interaction scores for complex structures of ACE2-spike-S1 and its mutants. E: Number of COVID-19 patients in Europe and USA.

    Figure  5.  Epidemic situation for spike-S1 mutants and receptor-ligand binding of spike-S1 mutants

    A: Number of amino acid mutants in spike-S1 at different time points. B: Distribution of spike-S1 mutants worldwide. C: Number of V354F and V470A mutants at different times. D: Optical density at 450 nm (OD450) for wild-type spike-S1 and its mutants, i.e., V354F and V470A mutants.

    Figure  6.  Different ACE2 sequences have different binding energies to spike-S1

    A: Sequence similarity between different ACE2 sequences. B: Phylogenetic tree for different ACE2 sequences. C: Sequence logo for ACE2. Red, yellow, and blue triangles represent ACE2 residues that could potentially form a hydrogen bond with the spike-S1 protein of SARS-CoV-2-Wuhan01 and an ionic or PiPistack interaction with the corresponding residues in spike-S1. D: Binding interaction energy scores for ACE2 and spike-S1.

    Table  1.   Molecular divergence among SARS-CoV-2 and its mutants

    dN/ds(ω)-H dN/ds(ω)-L dN/ds(ω)>>1 mutant
    Mpro 0/4.70E-3 (0) 0/4.70E-3 (0) N/A
    Papain-like protease 2.00E-3/3.00E-3 (0.67) 2.00E-4/7.00E-4 (0.29) N/A
    RNA-dependent RNA polymerase 5.00E-4/0 (N/A) 0/1.60E-3 (0) N/A
    Helicase 7.00E-4/0 (N/A) 0/2.30E-3 (0) N/A
    Spike-S1 7.00E-4/0 (N/A) 1.30E-3/2.1E-3(0.62) N/A
    Spike-S2 7.00E-4/0 (N/A) 0/2.50E-3 (0) N/A
    ORF3a 3.20E-3/0 (N/A) 0/5.10E-3 (0) N/A
    Envelope protein 6.10E-3/0 (0) N/A N/A
    Membrane glycoprotein 2.00E-3/6.10E-3(0.33) 0/1.23E-2 (0) N/A
    ORF6 protein N/A N/A N/A
    ORF7a protein 3.70E-03/0 (N/A) N/A N/A
    ORF8 protein 7.20E-3/0 (N/A) 0/1.22E-2 (0) N/A
    Nucleocapsid phosphoprotein 1.00E-3/0 (N/A) 0/3.40E-3 (0) N/A
    ORF10 protein 1.15E-2/0 (N/A) N/A N/A
    H and L represent highest dN/dS (ω) values for SARS mutants. N/A: Not available.
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  • 收稿日期:  2020-10-14
  • 录用日期:  2021-03-15
  • 网络出版日期:  2021-03-16
  • 刊出日期:  2021-03-18

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