Center for Genome Technology and Biomolecular Engineering, Columbia University, New York, NY 10027, USA.
Department of Chemical Engineering, Columbia University, New York, NY 10027, USA.
Viruses. 2022 Jun 28;14(7):1413. doi: 10.3390/v14071413.
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.
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且能抵抗变异株的口服药物。最近，我们报告了体外实验结果，验证了靶向 SARS-CoV-2 RNA 依赖性 RNA 聚合酶（RdRp）和校对外切酶（ExoN）的联合药物作为潜在 COVID-19 治疗药物的使用。为了使核苷酸类似物成为有效的 SARS-CoV-2 抑制剂，需要具备两个特性：由 RdRp 高效掺入以及对 ExoN 的切除有较大抗性。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，当它们连接到 3' RNA 末端时，其对 ExoN 切除的抗性。我们发现，没有 2'-和 3'-OH 基团的双脱氧核苷酸和其他核苷酸对 ExoN 切除的抗性最大，而那些同时具有 2'-和 3'-OH 基团的核苷酸则被有效地切除。我们还发现，核苷酸类似物中的 3'-OH 基团比对 ExoN 切除的 2'-OH 更关键。由于 Nsp14/10 中的功能重要序列在所有 SARS-CoV-2 变异株中高度保守，这些鉴定出的核苷酸类似物的结构特征为设计有效的 RdRp 抑制剂提供了宝贵的见解，这些抑制剂可以被 RdRp 高效掺入并对 ExoN 切除有较大抗性。这些新开发的 RdRp 终止子将是评估它们在细胞培养和动物模型中抑制 SARS-CoV-2 能力的候选物，也许可以与其他外切酶抑制剂结合使用以提高其整体效果。
