SARS-CoV-2 NSP13 解旋酶 ATP 活性抑制劑的篩選

Abstract

嚴重急性呼吸系統綜合症冠狀病毒-2（SARS-CoV-2）是造成 2019 年全球冠狀病毒病（COVID-19）的病原體，造成數百萬人死亡並對生活產生重大影響。病毒非結構蛋白 13（NSP13）解旋酶利用 ATP 水解的能量在 5' 至 3' 方向上解開雙鏈 DNA 或 RNA，由於其在病毒複製中的關鍵作用，已被確定為一個具有發展性的靶標。在這篇論文中，我表達純化了重組 SARS-CoV-2 解旋酶。通過使用孔雀石綠檢測磷酸鹽的 ATP 酶活性，確定了先前發現可抑制多種抗 SARS-CoV-2 靶標的內部 FDA 批准藥物或化學合成化合物和天然化合物對解旋酶的抑制活性。並採用分子對接技術闡明 SARS-CoV-2 解旋酶抑製劑的構效關係。此外，我還使用了 3109 個 FDA 核准的藥物庫、201 個動物用藥物、3200 個解旋酶標靶庫等，以虛擬方式篩選出解旋酶潛在抑製劑。測定了這些化合物的半抑制濃度 IC50，並通過對接程式對複雜結構進行了建模，以提供結構原理。綜合上述，本研究找出了一些可能的 SARS-CoV-2 解旋酶 ATP 水解活性的抑製劑，為對抗 COVID-19 提供了潛在的策略。

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the global coronavirus disease 2019 (COVID-19) pandemic, causing millions of people death and significant impact on life. The viral non-structural protein 13 (NSP13) helicase, which uses energy from NTP hydrolysis to unwind double-stranded DNA or RNA in a 5' to 3' direction, has been recognized as a promising target due to its essential role in viral replication. In this thesis, the recombinant SARS-CoV-2 helicase was expressed and purified. By assaying its ATPase activity using malachite green to detect phosphate, the inhibitory activities of the in house potential compounds and natural compounds previously found to inhibit several anti-SARS-CoV-2 targets were determined. Molecular docking was employed to elucidate the binding modes of the inhibitors against SARS-CoV-2 helicase. Furthermore, three compound libraries were used to virtually screen out helicase potential inhibitors. In conclusion, this study identifies promising inhibitors of SARS CoV-2 helicase, offering potential strategies for combating COVID-19.