2. Biological targets for SARS-CoV2
One key discovery in understanding the secrets of SARS-CoV2 infection involves virus structure, especially viral spike (S) protein, which facilitates viral entry into target cells by binding host-cell receptor and then by fusing viral and host membranes (Li, 2016). SARS-CoV2 specifically recognizes Angiotensin-Converting Enzyme 2 (ACE2) as the receptor binding domain (RDB) for its S protein to mediate viral entry and infection (Zhou et al . 2020b). Based on the fact that SARS-CoV2 engages the same receptor used by SARS-CoV to mediate infection, and that both virus share sequence similarities of 80% between their S proteins (Zhou et al . 2020b), it was suggested that they could act in a similar way. It has to be pointed out that the viral attachment to host cell membrane via ACE2 is the first of a multi-step process involved in coronavirus infection; indeed, after the ligation to ACE2, indispensable for infection, the next step is the priming of S protein by cellular proteases, which consists of S protein cleavage at the S1/S2 and the S2 site, which allows fusion of viral and cellular membranes proteases on the host cell (Letko et al . 2020; Chen et al . 2020). As in the case of SARS-CoV (Li et al . 2005a), the S1 subunit, which contains the RBD, directly interacts with the peptidase domain (PD) of ACE2 providing for tight and higher affinity binding between virus and the host cell. Based on the fact that RBD of SARS-CoV2 is the critical determinant of viral tropism and infectivity, it was demonstrated that its mutations could alter the affinity to the binding receptor, leading to increased viral load (Ouet al . 2020). In particular, three mutants of SARS-CoV2 RBD (V367F, W436R, and D364Y) are correlated to higher human ACE2 affinity, ensuing higher infectivity. This discovery provides insights into SARS-CoV2 evolution and highlights how an increased affinity for human ACE2 due to RDB mutations could further favor COVID-19 diffusion. Because ACE2 is the receptor that SARS-CoV2 uses to anchor host cell, it is obvious to speculate that its expression could be correlated to viral infection susceptibility. Therefore, scientific efforts are focused on the study of ACE2 localization, in order to identify the possible route of viral infection, spread and replication throughout the body. ACE2 expression in the lung is concentrated in a small population of type II pneumocytes, which also express other genes positively correlated to SARS-CoV2 reproduction and transmission (Zhao et al . 2020), suggesting that alveolar pneumocytes could be a potential site of entrance of this virus, and prove a possible explanation for rapid lung viral expansion and pulmonary manifestations typical of COVID-19 patients. If on one side, ACE2-expressing lung cells may be the main target cells for coronavirus infection, on the other, Hamming et al . 2004 have already reported that other organs express ACE2, maybe explaining why some COVID-19 patients also exhibit non-respiratory symptoms. According to the single-cell RNA sequencing (scRNA-seq) and protein datasets, apart from lung and type II alveolar cells, heart, esophagus, kidney, bladder, ileum, oral cavity and testes are the organs at risk due to higher ACE2 expression (Zou et al . 2020; Xuet al . 2020a). To date, in the attempt to find a potential drug against COVID-19, human recombinant soluble ACE2 (hrsACE2), which has already been tested in phase 1 and phase 2 clinical trials for ARDS and COVID-19 (Haschke et al . 2013; Khan et al . 2017; https://clinicaltrials.gov/ct2/show/NCT00886353), can reduce viral growth in Vero E6 cells, most probably by acting as a decoy receptor and preventing viral binding to the natural membrane-bound ACE2 (Monteilet al . 2020).
Beyond ACE2, it was recently found that SARS-CoV2 also uses the cellular transmembrane protease serine 2 (TMPRSS2) for S protein priming, another key event for virus entrance into host cell (Hoffmann et al., 2020). TMPRSS2 is a cell surface protein of the serine protease transmembrane family type II that is broadly expressed by epithelial cells (Zouet al . 2020; Xu et al . 2020a) and is involved in the cleavage of the SARS-CoV and influenza virus hemagglutinin protein (Böttcher et al . 2006). As other member of its family, TMPRSS2 favors the entry of the virus into the lungs leading to respiratory infections (Shulla et al . 2011). This protease was already described by Gierer et al . (2013) and Matsuyama et al . (2010) as the enzyme responsible for SARS-CoV infection. Hoffman et al., (2020) found that, SARS-CoV2 uses both TMPRSS2 and endosomal cysteine proteases cathepsin B and L (CatB/L) to enter host cells. The inhibition of TMPRSS2 by means of Camostat mesylate, an TMPRSS2 inhibitor, partially blocked SARS-CoV2 entry, suggesting CatB/L involvement (Kawaseet al. 2012). Moreover, the same authors found that co-treatment with Camostat mesylate and E-64d, an inhibitor of CatB/L, completely abrogated virus entry in the same cells, indicating that the virus can use both CatB/L as well as TMPRSS2 for S protein priming in these cell lines. In contrast, the sole Camostat mesylate was not able to block SARS-CoV-2 entry into the TMPRSS2 knock-down 293T cells, confirming that the S protein of SARS-CoV-2 could employ TMPRSS2 for its priming.
Other lines of research are focusing their attention on the coronavirus 3-chymotrypsin-like protease (3CLpro), also known as Mpro, a cysteine protease present in the Coronavirus replicase polyprotein (Zhou et al . 2019). This protease plays a critical role both in the immune regulation and in viral replication in that it regulates the proteolytic cleavage of some polyprotein. 3CLpro drives the cleavage of polyproteins pp1a and pp1ab, which in turn are responsible for the generation of functional proteins such as RNA polymerase, endoribonuclease and exoribonuclease (Khan et al . 2020). For this reason, it was speculated that 3CLpro could represent an attractive target for COVID-19 treatment. In this context, two different molecular docking and molecular dynamic simulation studies reveled 4 drugs that could act against 3CLpro: the antibacterial drug talampicillin, the antipsychotic drug lurasidone (Elmezayen et al. 2020), and the antiviral drug raltegravir and paritaprevir, which were already used in the antiretroviral therapy against the Human Immuno-deficiency Virus (HIV) infections as integrase strand transfer inhibitors (INSTI) (Khanet al . 2020). 3CLpro also cleaves the 2’-O-Ribose Methyltransferase (2’-O-MTase), a protein that catalyzes the methylation of 5’-terminal cap structure of viral mRNAs (Chen et al . 2011). Because this reaction is crucial for viral replication and expression in host cells (Menachery et al . 2014), 2’-OMTase was suggested as another possible druggable target for COVID-19 treatment (Khan et al . 2020), although it is still unclear whether 2’-O-MTase, as well as 3CLpro, contributes to SARS-CoV2 infection.