Li Yan, Zhang Hui-Xia, Luo Wen-Di, Lam Christopher Wai Kei, Wang Cai-Yun, Bai Li-Ping, Wong Vincent Kam Wai, Zhang Wei, Jiang Zhi-Hong
State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Guangdong-Hong Kong-Macao Joint Laboratory of Respiratory Infectious Disease (Macau University of Science and Technology), Taipa, Macau, China.
Faculty of Medicine and State Key Laboratory of Quality Research in Chinese Medicines, Macau University of Science and Technology, Taipa, Macau, China.
Front Pharmacol. 2021 May 4;12:647280. doi: 10.3389/fphar.2021.647280. eCollection 2021.
Remdesivir (RDV) has generated much anticipation for its moderate effect in treating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, the unsatisfactory survival rates of hospitalized patients limit its application to the treatment of coronavirus disease 2019 (COVID-19). Therefore, improvement of antiviral efficacy of RDV is urgently needed. As a typical nucleotide analog, the activation of RDV to bioactive triphosphate will affect the biosynthesis of endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs), which are essential to RNA and DNA replication in host cells. The imbalance of RN pools will inhibit virus replication as well. In order to investigate the effects of RDV on cellular nucleotide pools and on RNA transcription and DNA replication, cellular RNs and dRNs concentrations were measured by the liquid chromatography-mass spectrometry method, and the synthesis of RNA and DNA was monitored using click chemistry. The results showed that the IC values for BEAS-2B cells at exposure durations of 48 and 72 h were 25.3 ± 2.6 and 9.6 ± 0.7 μM, respectively. Ten (10) μM RDV caused BEAS-2B arrest at S-phase and significant suppression of RNA and DNA synthesis after treatment for 24 h. In addition, a general increase in the abundance of nucleotides and an increase of specific nucleotides more than 2 folds were observed. However, the variation of pyrimidine ribonucleotides was relatively slight or even absent, resulting in an obvious imbalance between purine and pyrimidine ribonucleotides. Interestingly, the very marked disequilibrium between cytidine triphosphate (CTP) and cytidine monophosphate might result from the inhibition of CTP synthase. Due to nucleotides which are also precursors for the synthesis of viral nucleic acids, the perturbation of nucleotide pools would block viral RNA replication. Considering the metabolic vulnerability of endogenous nucleotides, exacerbating the imbalance of nucleotide pools imparts great promise to enhance the efficacy of RDV, which possibly has special implications for treatment of COVID-19.
瑞德西韦（RDV）因其在治疗严重急性呼吸综合征冠状病毒2（SARS-CoV-2）感染方面的适度疗效而备受期待。然而，住院患者不尽人意的生存率限制了其在2019冠状病毒病（COVID-19）治疗中的应用。因此，迫切需要提高瑞德西韦的抗病毒疗效。作为一种典型的核苷酸类似物，瑞德西韦活化为生物活性三磷酸形式会影响内源性核糖核苷酸（RNs）和脱氧核糖核苷酸（dRNs）的生物合成，而这两者对于宿主细胞中的RNA和DNA复制至关重要。核糖核苷酸库的失衡也会抑制病毒复制。为了研究瑞德西韦对细胞核苷酸库以及RNA转录和DNA复制的影响，采用液相色谱-质谱法测量细胞核糖核苷酸和脱氧核糖核苷酸浓度，并使用点击化学监测RNA和DNA的合成。结果显示，BEAS-2B细胞在暴露48小时和72小时时的半数抑制浓度（IC）分别为25.3±2.6μM和9.6±0.7μM。10μM瑞德西韦处理24小时后导致BEAS-2B细胞停滞于S期，并显著抑制RNA和DNA合成。此外，观察到核苷酸丰度普遍增加，且特定核苷酸增加超过2倍。然而，嘧啶核糖核苷酸的变化相对较小甚至没有变化，导致嘌呤和嘧啶核糖核苷酸之间明显失衡。有趣的是，三磷酸胞苷（CTP）和一磷酸胞苷之间非常明显的不平衡可能是由于CTP合酶受到抑制所致。由于核苷酸也是病毒核酸合成的前体，核苷酸库的扰动会阻断病毒RNA复制。考虑到内源性核苷酸的代谢脆弱性，加剧核苷酸库的失衡有望提高瑞德西韦的疗效，这可能对COVID-19的治疗具有特殊意义。
