Identifying Structural Features of Nucleotide Analogues to Overcome SARS-CoV-2 Exonuclease Activity.
Identifying Structural Features of Nucleotide Analogues to Overcome SARS-CoV-2 Exonuclease Activity.
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DOI：
10.3390/v14071413
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发表时间：
2022-06-28
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随着新的SARS-CoV-2变异株在全球范围内的传播，仍然迫切需要开发有效的和抗变异的口服药物。最近，我们报告了体外结果，验证了靶向SARS-CoV-2 RNA依赖性RNA聚合酶（RdRp）和校正核酸外切酶（ExoN）的联合药物作为潜在COVID-19治疗剂的使用。要使核苷酸类似物成为有效的SARS-CoV-2抑制剂，需要两个性质：RdRp的有效掺入和对ExoN切除的实质性抗性。在这里，我们选择并评估了具有各种结构特征的核苷酸类似物，当它们连接在3′ RNA末端时，它们对ExoN去除具有抗性。我们发现，双脱氧核苷酸和其他缺乏2′-和3′-OH基团的核苷酸对ExoN切除的抵抗力最强，而那些同时具有2′-和3′-OH基团的核苷酸则被有效地去除。我们还发现，核苷酸类似物中的3′-OH基团比2′-OH基团对于ExoN的切除更关键。由于Nsp 14/10中功能上重要的序列在所有SARS-CoV-2变体中高度保守，这些核苷酸类似物的鉴定结构特征为设计有效的RdRp抑制剂提供了宝贵的见解，这些抑制剂可以同时有效地被RdRp掺入并基本上抵抗ExoN切除。这种新开发的RdRp终止子将是评估其在细胞培养和动物模型中抑制SARS-CoV-2的能力的良好候选物，也许与额外的核酸外切酶抑制剂组合以增加其整体有效性。
With the recent global spread of new SARS-CoV-2 variants, there remains an urgent need to develop effective and variant-resistant oral drugs. Recently, we reported in vitro results validating the use of combination drugs targeting both the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) and proofreading exonuclease (ExoN) as potential COVID-19 therapeutics. For the nucleotide analogues to be efficient SARS-CoV-2 inhibitors, two properties are required: efficient incorporation by RdRp and substantial resistance to excision by ExoN. Here, we have selected and evaluated nucleotide analogues with a variety of structural features for resistance to ExoN removal when they are attached at the 3′ RNA terminus. We found that dideoxynucleotides and other nucleotides lacking both 2′- and 3′-OH groups were most resistant to ExoN excision, whereas those possessing both 2′- and 3′-OH groups were efficiently removed. We also found that the 3′-OH group in the nucleotide analogues was more critical than the 2′-OH for excision by ExoN. Since the functionally important sequences in Nsp14/10 are highly conserved among all SARS-CoV-2 variants, these identified structural features of nucleotide analogues offer invaluable insights for designing effective RdRp inhibitors that can be simultaneously efficiently incorporated by the RdRp and substantially resist ExoN excision. Such newly developed RdRp terminators would be good candidates to evaluate their ability to inhibit SARS-CoV-2 in cell culture and animal models, perhaps combined with additional exonuclease inhibitors to increase their overall effectiveness.
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