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.