2.1. Spike Protein
The coronavirus structural proteins include the spike glycoprotein (S-protein), envelope protein (E-protein), membrane protein (M-protein), and the nucleocapsid protein (N-protein). The S-protein of SARS‐CoV‐2 is similar to the coronavirus strain SARS-CoV with over 72% amino acid sequence similarity (Chen, Guo, Pan & Zhao, 2020). SARS‐CoV‐2 can bind to angiotensin converting enzyme 2 (ACE2) through the receptor-binding domain (RBD) of the S-protein (Lan et al., 2020) with a higher affinity relative to SARS‐CoV (Chen, Guo, Pan & Zhao, 2020). In addition, SARS‐CoV‐2 does not use other receptors as other coronavirus, such as aminopeptidase N and dipeptidyl peptidase‐4 (Zhou et al., 2020a) .
The S-protein of SARS-CoV-2 consists of two subunits: a globular S1 domain at the N-terminal region and the membrane-proximal S2 domain. The RBD within S1 subunit is essential for the virus attachment to host cell receptor ACE2; while S2 is critical for virus entry by regulating viral membrane fusion to host cell membrane (Kirchdoerfer et al., 2016; Wrapp et al., 2020; Yan, Zhang, Li, Xia, Guo & Zhou, 2020). Both S1-RBD and S2 domains represent important potential targets for the development of SARS-CoV-2 vaccine and therapeutic drugs (Huang et al., 2020a). In fact, many of the currently developed vaccines target S-protein of SARS-CoV-2 (Folegatti et al., 2020; Logunov et al., 2020; Smith et al., 2020; Zhu et al., 2020). RBD seems to be a good target for SARS-CoV-2 vaccine, since a recombinant RBD protein of SARS-CoV-2 prepared from insect cells was reported to induce serum antibodies that could bind the RBD and neutralize viral infection in nonhuman primates (Walsh et al., 2020; Yang et al., 2020). The development of monoclonal antibody against or inhibitors of S-protein might hold great promise to find therapeutic candidates for COVID-19.
ACE2
The S-protein of both SARS-CoV and SARS-CoV- has been known to utilize ACE2 as a receptor for host cell entry (Li et al., 2003; Wang et al., 2020a). ACE2 is a metallopeptidase that is expressed on major viral target cells, such as type II pneumocytes and enterocytes (Hamming, Timens, Bulthuis, Lely, Navis & van Goor, 2004; Mossel et al., 2008; To & Lo, 2004). ACE2 is expressed not only in type II alveolar cells (AT2), but also in myocardial cells, proximal tubule cells of the kidney, ileum and esophagus epithelial cells, and bladder urothelial cells, and as well as in brain cells, establishing the basis for possible extrapulmonary invasion of SARS-CoV-2.
The catalytic domain of ACE2 binds to S-protein with high affinity (Li et al., 2003; Wong, Li, Moore, Choe & Farzan, 2004). Binding of S-protein to ACE2 triggers conformational rearrangements in S-protein, which are believed to increase the sensitivity of the S-protein to proteolytic enzymes at the border between the S1 and S2 subunits for priming to facilitate the viral entry into the cells.
A clinical-grade soluble recombinant human ACE2 protein (rhACE2) was shown to inhibit attachment of SARS-CoV-2 to simian Vero-E6 cells and prevent SARS-CoV-2 infection of engineered human capillary organoids and kidney organoids (Vero-E 6 cells) (Monteil et al., 2021). However, whether rhACE2 administration in vivo can prevent SARS-CoV-2 infection remains unknown. The roles of ACE2 inhibitors, such as nicotinamide (Takahashi, Yoshiya, Yoshizawa-Kumagaye & Sugiyama, 2015), in SARS-CoV-2 infection should be further investigated.
S-protein can also regulate cellular signing pathways. S-protein has been shown to activate the mitogen-activated protein kinase (MEK)/ extracellular signal-regulated kinase (ERK) pathway and increase the downstream chemokine expression through ACE2 (Chen et al., 2010). ANG II can also reduce the expression of ACE2 mRNA and increase the extracellular signal-regulated kinase (ERK) 1/ERK2 activity, which was prevented by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 in vascular smooth muscle cells (Gallagher, Ferrario & Tallant, 2008). Further study indicates that MAPK kinase (MEK) inhibitors (VS-6766, trametinib and selumetinib) reduced ACE2 expression and attenuated the release of inflammatory cytokines during SARS-Cov-2 infection (Zhou et al., 2020b).
2.3. Transmembrane Serine Protease 2 (TMPRSS2)
TMPRSS2, a transmembrane serine protease in airway epithelial cells and alveolar cells, plays a critical role in viral entry into host cells. Like ACE2, TMPRSS2 also express in the heart, digestive tract, liver, kidney, brain, and other organs (Dong et al., 2020).
One key function of TMPRSS2 is to prime the viral S-protein to facilitate the interaction between S-protein and ACE2, which is essential for viral infectivity. Both SARS-CoV and SARS-CoV-2 use the serine protease TMPRSS2 for S protein priming (Hoffmann et al., 2020) . The binding of RBD within the S1 domain to ACE2 could trigger the effects of TMPRSS2 on the cleavage of S-protein at the S1 and S2 border sites and facilitate cell membrane fusion for viral entry (Walls, Park, Tortorici, Wall, McGuire & Veesler, 2020).
Inhibition of TMPRSS2 has been shown to block the entry of SARS-CoV-2 (Hoffmann et al., 2020; Sagar et al., 2021). Inhibition of TMPRSS2 by camostat mesylate in human lung Calu-3 cells significantly reduced infection of the cells by SARS-CoV-2. Nafamostat mesylate, which has been demonstrated to inhibit TMPRSS2-dependent host cell entry of MERS-CoV (Yamamoto et al., 2016), can also prevent SARS-CoV-2 entry into host cells with roughly 15-fold-higher efficiency than camostat mesylate (Hoffmann et al., 2020). Another TMPRSS2 inhibitor, α-1antitrypsin, blocks the SARS-CoV-2 from entering host cells. The clinical efficacy of these inhibitors is under evaluation for COVID-19.