Inhibition of TMPRSS2 and 3CL Proteases by natural polyphenols to prevent SARS-CoV-2 infectivity and replication

在这项研究中，我们建议证明多酚(Pls)可以通过抑制SARS-CoV-2病毒感染和复制的两个关键蛋白酶来抑制SARS-CoV-2病毒的有害影响。要感染细胞，SARS-CoV-2病毒必须进入宿主细胞，并与其膜受体ACE-2结合。参与S蛋白激活和病毒初始进入的主要宿主酶是跨膜丝氨酸蛋白酶2(TMPRSS2)。一旦进入细胞，SARS-CoV-2的一段片段就会被3-胰凝乳酶样半胱氨酸蛋白酶(3CL)切割，该酶控制冠状病毒的复制，对其生命周期至关重要。我们的假设是，某些PLS可以通过两个水平作用来减弱病毒的作用，例如：1)阻止TMPRSS2激活蛋白-S，2)抑制冠状病毒复制和成熟所需的3CL。为此，我们提出了3个目标：1-使用多学科的方法，理论(分子建模和分子对接)和实验(DLS，SPR，颗粒跟踪)，以更好地了解酚类配体与TMPRSS2和3CL之间的物理化学相互作用；2-通过测量在存在或不存在pls浓度增加的情况下，TMPRSS2和3CL对多肽序列的切割来评价pls对蛋白酶的抑制作用；3-通过TMPRSS2促进的蛋白-S病毒与血管紧张素转换酶-2之间的相互作用，以及在培养的细胞模型(肺、血脑屏障的血管内皮细胞)上的相互作用，评估PLS对病毒内化的影响。此外，随着SARS-CoV-2入侵神经系统从而影响大脑的证据的积累，我们建议研究pls对背根节细胞系、参与神经炎症的神经元和小胶质细胞的影响，因为一旦进入大脑，SARS-CoV-2和SARS-CoV一样，可以诱导神经炎症。本研究的结果将证明PLS对SARS-CoV-2的保护作用，从而为SARS-CoV-2的治疗方法和神经侵袭机制的发展开辟了多个视角。

In this research, we propose to prove that polyphenols (PLs) can inhibit the harmful effects of the SARS-CoV-2 virus by inhibiting two key proteases for virus infection and replication. To infect a cell, the SARS-CoV-2 virus must enter a host cell and bind to its membrane receptor ACE-2. The main host protease involved in the activation of protein -S on primary target cells and initial viral entry is the transmembrane serine protease 2 (TMPRSS2). Once in the cell, a fragment of SARS-CoV-2 is cleaved by the 3-chymotrypsin-like cysteine protease (3CL), which controls the replication of the coronavirus, essential for its life cycle. Our hypothesis is that certain PLs could attenuate the effects of the virus by acting on two levels, e.g.: 1) preventing TMPRSS2 from activating the protein -S, and 2) inhibiting the 3CL necessary for the replication and maturation of coronavirus. To do this, we propose 3 objectives: 1- Use a multidisciplinary approach, theoretical (molecular modeling and molecular docking) and experimental (DLS, SPR, particle tracking), to better understand the physicochemical interactions between phenolic ligands and TMPRSS2 and 3CL; 2- In vitro evaluation of protease inhibition by PLs by measuring the cleavage of peptide sequences by TMPRSS2 and 3CL in the presence, or absence, of increasing concentrations of PLs; 3-To assess the effects of PLs on the internalization of viruses mediated by the interaction between the protein -S viral and ACE-2, facilitated by the TMPRSS2, as well as the interactions on cellular models in culture (pulmonary, vascular endothelial cells of the blood-brain-barrier). In addition, as the evidence accumulates on the invasion of SARS-CoV-2 in the nervous system and thus it effects on the brain, we propose to study the effects of PLs on the dorsal root ganglia cell line, on neuronal and microglial cells involved in neuroinflammation because once in the brain, SARS-CoV-2, like SARS-CoV, can induce neuroinflammation. The results of this study will demonstrate the protective potential of PLs against SARS-CoV-2, hence, open up multiple perspectives in the development of treatments for SARS-CoV-2 and in the neuroinvasive mechanisms.