製備活化的TMPRSS2胞外域以測試SARS-CoV-2進入細胞的抑制劑

Abstract

由嚴重急性呼吸道症候群冠狀病毒2型 (SARS-CoV-2) 引起的 2019 年冠狀病毒病 (COVID-19) 已在全球奪去許多人的生命，且仍在蔓延中。 病毒在與宿主細胞表面的血管緊張素轉化酶 2 (ACE2) 受體結合後，細胞表面的跨膜絲胺酸蛋白酶2 (TMPRSS2) 會切割 SARS-CoV-2 刺突蛋白，使病毒與細胞膜融合並進入細胞。 因此，已知藥物（如 camostat 和 nafamostat），藉著不可逆地抑制 TMPRSS2，從而抑制病毒進入。 如本文所述，我使用桿狀病毒感染的昆蟲細胞表達並激活TMPRSS2 胞外域以測定我們實驗室合成的抑制劑以及選定的天然產物和 FDA 批准的藥物。 這些化合物還使用弗林蛋白酶 (Furin) 和組織蛋白酶 L (Cathepsin L) 的試劑進行了分析，Furin 是一種人類細胞表面絲氨酸蛋白酶，可以與 TMPRSS2 分別在刺突蛋白的 S1/S2 及 S2' 位點切割。Cathepsin L一種參與切割刺突蛋白的細胞內蛋白酶，從而由胞內體釋放入侵的病毒。 根據 IC50 建立針對 TMPRSS2、Furin 和/或 Cathepsin L的抑制劑的構效關係，並通過分子嵌合計算 (docking) 對結構進行模擬以提供抑制的解釋。因此，這項研究提供了可能的 TMPRSS2、Furin 和/或 Cathepsin L 抑制劑來阻止 SARS-CoV-2 進入細胞。

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in global fatalities and continues to spread. Transmembrane protease, serine 2 (TMPRSS2) on the surface of infected host cells cleaves SARS-CoV-2 spike protein, enabling the primed virus to fuse with the cell membrane and enter the cells after binding with the angiotensin-converting enzyme 2 (ACE2) receptor. Therefore, known antivirals like camostat and nafamostat irreversibly inhibit TMPRSS2 and virus entry. In this thesis, TMPRSS2 ectodomain was expressed using baculovirus-infected insect cells and activated to screen the inhibitors synthesized in our laboratory, as well as the selected natural products and FDA-approved drugs. These compounds were also assayed using the commercial kits to determine their inhibitory effects against Furin and Cathepsin L. Furin is a human cell-surface serine protease that works with TMPRSS2 to cleave the spike protein at the canonical S1/S2 and S2' site, respectively. Cathepsin L is an intracellular protease involved in cleaving the spike protein to release the invading virus from the endosome. Structure-activity relationships of the inhibitors against TMPRSS2, Furin, and/or Cathepsin L were established from the IC50 and the complex structures were modeled by the docking program to provide the structural rationale. Therefore, this study offers promising TMPRSS2, Furin, and/or Cathepsin L inhibitors to block SARS-CoV-2 entry.