The significant number of deaths and infection caused by the new coronavirus SARS-CoV-2 has created an urgent demand for effective and readily available drugs for the treatment of COVID-19. However, the requirements for biosafety level 3 (NB-3) laboratories for experiments with the virus has made it very challenging for such research to meet this demand. It is known that angiotensin-converting enzyme 2 (ACE2), located on the surface of host cells, serves as the viral receptor for the spike (S) protein of SARS-CoV-2. This protein is a tetramer subdivided into S1 and S2 regions, with the former containing the receptor-binding domain (RBD). Therefore, drugs that interfere with the interaction between the spike and the receptor (as well as accessory proteins) or suppress their expression could inhibit the entry and spread of SARS-CoV-2 between cells. In this context, we standardized the use of recombinant SARS-CoV-2 S1 Protein with hFc (human Fc) for the analysis of binding in VERO E6 cells by flow cytometry, aiming to provide a new tool for identifying drugs and neutralizing antibodies, thus eliminating the need for NB-3 laboratories. Because minocycline (MCL), nimesulide (NMS), and berberine (BBR) have effects related to the ACE2 receptor, inhibit inflammation, and do not suppress the adaptive immune response (crucial for patient recovery), we investigated whether these drugs prevent the absorption of the spike protein into the host cell. For this purpose, we used VERO E6 cells under control conditions, pre-treated with these drugs and exposed to recombinant SARS-CoV-2 S1 Protein with hFC. We found that an exposure time of 30 min and a concentration of 10 μg/mL of spike S1 caused a strong signal detected by flow cytometry, using the secondary anti-hFc antibody conjugated with Alexa Fluor 647. Pre-treatment of cells with BBR for 30 min suppressed the signal from spike-positive cells, suggesting that this alkaloid interferes with spike adsorption on ACE2. The pre-incubation of spike protein with BBR did not alter its adsorption and internalization, indicating that BBR does not directly interact with spike protein. The ACE2 inactivation with a specific antibody inhibited spike protein adsorption and internalization. Furthermore, the pharmacological treatments did not alter the expression of ACE2. Exposure to spike protein increased IFNγ levels and the treatments with MCL and NMS were effective in inhibiting this increase. Taken together, we standardized a technique for analyzing the adsorption of SARS-CoV-2 and studying molecules that inhibit this process. Additionally, we demonstrated that BBR blocks spike entry bypre-binding to the host cell,and that the ACE2 receptor inactivation prevents Spike protein adsorption and penetration into cells.

新型冠状病毒 SARS-CoV-2 造成大量死亡和感染，迫切需要有效且易于获得的药物来治疗 COVID-19。然而，对生物安全3级（NB-3）实验室进行病毒实验的要求使得此类研究满足这一需求非常具有挑战性。众所周知，血管紧张素转换酶 2 (ACE2) 位于宿主细胞表面，是 SARS-CoV-2 刺突 (S) 蛋白的病毒受体。该蛋白是一个四聚体，分为 S1 和 S2 区域，其中前者包含受体结合结构域 (RBD)。因此，干扰刺突与受体（以及辅助蛋白）之间相互作用或抑制其表达的药物可以抑制 SARS-CoV-2 在细胞间的进入和传播。在此背景下，我们标准化使用重组SARS-CoV-2 S1蛋白与hFc（人Fc）通过流式细胞术分析VERO E6细胞中的结合，旨在为识别药物和中和抗体提供新工具，从而消除对 NB-3 实验室的需求。由于米诺环素 (MCL)、尼美舒利 (NMS) 和小檗碱 (BBR) 具有与 ACE2 受体相关的作用，可抑制炎症，并且不会抑制适应性免疫反应（对患者康复至关重要），因此我们研究了这些药物是否会阻止吸收刺突蛋白进入宿主细胞。为此，我们在对照条件下使用了 VERO E6 细胞，用这些药物进行预处理，并暴露于含有 hFC 的重组 SARS-CoV-2 S1 蛋白。我们发现，使用与 Alexa Fluor 647 偶联的抗 hFc 二抗，暴露时间为 30 分钟且尖峰 S1 浓度为 10 μg/mL 会导致流式细胞仪检测到强信号。 30 分钟抑制了来自尖峰阳性细胞的信号，表明该生物碱干扰了 ACE2 上的尖峰吸附。刺突蛋白与 BBR 预孵育并没有改变其吸附和内化，表明 BBR 不直接与刺突蛋白相互作用。用特异性抗体灭活 ACE2 可抑制刺突蛋白的吸附和内化。此外，药物治疗并没有改变ACE2的表达。暴露于刺突蛋白会增加 IFNγ 水平，而 MCL 和 NMS 治疗可有效抑制这种增加。总之，我们标准化了一种分析 SARS-CoV-2 吸附并研究抑制这一过程的分子的技术。此外，我们证明 BBR 通过与宿主细胞预结合来阻止 Spike 进入，并且 ACE2 受体失活可阻止 Spike 蛋白吸附和渗透到细胞中。