SARS-CoV-2 Spike受體結合域與人血管緊張素轉換酶2的相互作用和抑制

Abstract

在2019年，嚴重急性呼吸系統綜合症-冠狀病毒-2（SARS-CoV-2）引起了繼2003年SARS事件後，另一場全球性的疾病大流行（COVID-19），並奪走了很多人的性命。病毒表面的刺突蛋白通過其受體結合域（RBD）與人血管緊張素轉換酶2（ACE2）結合之後，造成宿主感染。破壞RBD和ACE2的相互作用可以阻止病毒的進入，來達致預防感染的效果，這是抗COVID-19的有用策略。在此論文中，我使用受桿狀病毒感染的昆蟲細胞表達含有N-端8個His標籤的重組Delta及 Omicron BA.5 RBD和ACE2，並利用Ni-NTA親和層析柱純化。接下來，利用BIAcore實驗鑑定出它們的相互作用，確定了delta及omicron BA.5 RBD:ACE2 KD 分別為 81.28 及 18.83 nM，結果顯示它們具有非常緊密的結合。在蛋白質-蛋白質相互作用抑制劑如合成化合物或天然產物的存在下，RBD:ACE2複合物會被解離。而熱位移測定實驗進一步確定了RBD是抑制劑的直接靶標。由於針對RBD的治療性抗體價格昂貴，我們發現的RBD：ACE2小分子抑制劑可以作為開發抗COVID-19藥物的起點。

In 2019, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) led to another global pandemic, known as COVID-19, following the 2003 SARS outbreak, claiming numerous lives. For infection, the Spike protein on the surface of virus needs to bind with the human angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD). Disrupting the interaction of RBD and ACE2 could prevent the infection and represents a useful strategy for anti-COVID-19. In this thesis, baculovirus-infected insect cells were used to express the recombinant delta and omicron BA.5 RBD and ACE2 containing N-terminal His-tag and purified with Ni-NTA affinity column. Their interaction was characterized using BIAcore experiments to determine delta and omicron BA.5 RBD:ACE2 dissociation constants KD of 81.28 and 18.83 nM, respectively, a very tight binding. In the presence of protein-protein interaction inhibitors such as synthesized compounds or natural products, the complex was dissociated. The thermal shift experiments further identified the direct target of the inhibitors to be RBD. Since therapeutic antibodies against RBD are expensive, our discovery of small-molecule inhibitors of RBD:ACE2 could serve as a starting point for developing anti-COVID-19 drugs.