Department of Intensive Care Medicine, School of Medicine, Pontificia Universidad Católica de Chile, Santiago/8330024, Chile.
Biomed Res Int. 2021 Jul 23;2021:6614000. doi: 10.1155/2021/6614000. eCollection 2021.
Chloroquine (CQ) and hydroxychloroquine (HCQ) have shown the ability to inhibit viral replications of coronaviridae viruses such as SARS-CoV and SARS-CoV-2. However, clinical trial outcomes have been disparate, suggesting that CQ and HCQ antiviral mechanisms are not fully understood. Based on three-dimensional structural similarities between HCQ and the known ACE2 specific inhibitor MLN-4760, we compared their modulation on ACE2 activity. Here we describe, for the first time, in a cell-free system that HCQ directly and dose-dependently inhibits the activity of recombinant human ACE2, with a potency similar to the MLN-4760. Further analysis suggests that HCQ binds to a noncompetitive site other than the one occupied by MLN-4760. We also determined that the viral spike glycoprotein segment that comprises the RBD segment has no effect on ACE2 activity but unexpectedly was able to partially reverse the inhibition induced by HCQ but not that by MLN-4760. In summary, here we demonstrate the direct inhibitory action of HCQ over the activity of the enzyme ACE2. Then, by determining the activity of ACE2, we reveal that the interaction with the spike protein of SARS-CoV-2 leads to structural changes that at least partially displace the interaction of the said enzyme with HCQ. These results may help to explain why the effectiveness of HCQ in clinical trials has been so variable. Additionally, this knowledge could be used for to develop techniques for the detection of SARS-CoV-2.
氯喹（CQ）和羟氯喹（HCQ）已显示出抑制冠状病毒科病毒（如 SARS-CoV 和 SARS-CoV-2）复制的能力。然而，临床试验结果却大相径庭，这表明 CQ 和 HCQ 的抗病毒机制尚未完全阐明。基于 HCQ 与已知的 ACE2 特异性抑制剂 MLN-4760 之间的三维结构相似性，我们比较了它们对 ACE2 活性的调节作用。在这里，我们首次在无细胞系统中描述了 HCQ 直接且剂量依赖性地抑制重组人 ACE2 活性的情况，其效价与 MLN-4760 相似。进一步的分析表明，HCQ 结合在一个不同于 MLN-4760 占据的非竞争性位点。我们还确定了构成 RBD 片段的病毒刺突糖蛋白片段对 ACE2 活性没有影响，但出人意料的是，它能够部分逆转 HCQ 诱导的抑制作用，但不能逆转 MLN-4760 诱导的抑制作用。总之，在这里我们证明了 HCQ 对 ACE2 酶活性的直接抑制作用。然后，通过确定 ACE2 的活性，我们揭示了与 SARS-CoV-2 的刺突蛋白相互作用导致结构发生变化，至少部分取代了所述酶与 HCQ 的相互作用。这些结果可能有助于解释为什么 HCQ 在临床试验中的有效性如此不同。此外，这些知识可用于开发用于检测 SARS-CoV-2 的技术。
