Overview of Targets and Potential Drugs of SARS-CoV‑2 According to the Viral Replication

INTRODUCTION

In late December 2019 in Wuhan, China, several patients were diagnosed with viral pneumonia. The virus, now known as SARS-CoV-2, is characterized by its strong pathogenicity. Since the outbreak of this global pandemic disease, people around the world have been severely affected, and this pandemic around the world is extremely grim. Real-time data from the World Health Organization has shown that as of 13 November 2020, the number of global infections reached 52 177 708, and the global death toll reached 1 286 063.

Therefore, finding new therapeutic targets and developing effective drugs is required for addressing this pandemic. Currently, curative drugs for SARS-CoV-2 mainly include immune-related drugs and antiviral drugs. It is well documented that SARS-CoV-2 mainly consists of the S protein, E protein, M protein, N protein, and genome.

The N protein bends in a spiral shape to bind to the genome, while the S protein, E protein, and M protein bind to the viral envelope, and the S protein plays a recognition role when the virus enters the cell.1 These proteins are very crucial for the viral replication cycle (Figure 1), and inhibiting them may block the viral replication cycle as well as treat COVID-19.

Therefore, we summarize the potential targets and therapeutic drugs for the replication cycle of SARS-CoV-2, hoping to provide a reference for scientists and help to develop more drugs suitable for clinical treatment as soon as possible.

ADSORPTION

The adsorption of SARS-CoV-2 to the host cells is transmitted by the S protein of SARS-CoV-2 and ACE2 or CD147 receptors on the host cell. The core domain of S protein RBD can bind with ACE2 receptors, and this process exposes the fusion site of S2, which mediates the virus entering host cells with the help of proteases such as furin and TMPRSS2.

Spike Protein

Spike protein in coronavirus plays an important role in the process of virus infecting host cells, and it mediates the attachment, fusion, and entering host cells. Spike protein consists of three parts: the large outer membrane domain, the transmembrane domain, and the intracellular tail.

The receptor-binding subunits S1 subunit and S2 subunit are all located in the outland region, and they exist in two different structural conformations. S1 subunit binds to a receptor on the host cells and the virus attaches to them.

Then the conformation transforms, and the membrane fusion occurs via the S2 subunit.1 As for SARS-CoV-2, its S1 subunit protein can prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters.6 Recently, a molecular docking study has found that ivermectin, an FDA-approved broad-spectrum antiparasitic drug, can bind to the RBD region of SARS-CoV-2, thereby inhibiting its coupling to human ACE2 receptor.7

Moreover, Nitazoxanide, an antiprotozoal drug, can inhibit SARS-CoV-2 binding to ACE2 by interfering with the high glycosylated spike proteins of SARS-CoV-2, so it inhibits the entry of the virus into its target cells.8 Also, one study using surface plasmon resonance found that heparin binds to SARS- CoV-2 spike proteins, which suggests that heparin may be a potential inhibitor for viral entry.9

Arbidol (ARB)/umifenovir is an indole-derivative molecule that functions as a virus-host contains an N-terminal domain (NTD) and a C-terminal domain (CTD), which is also known as a receptor-binding domain (RBD). Recently, RBD in SARS-CoV-2 was said to be a key region to interact with receptor ACE2 in humans.2

RBD is composed of a core and a receptor-binding motif (RBM), and the RBM mediates the binding between virus and ACE2. SARS-CoV-2 RBD exposes less than SARS-CoV RBD, but its binding affinity is more potent, suggesting that the structure of SARS-CoV-2 RBM strengthen it, so it can be hypothesized that RBM monoclonal antibodies can inhibit the virus from binding to ACE2.3

At present, it has been found that recombinant SARS-CoV-2 RBD fragment can bind to ACE2 and prevent the virus from entering ACE2 target cells. Besides, the SARS-CoV RBD antibody can bind to the RBD of SARS- CoV-2, and the binding force of SARS-CoV-2 RBD is stronger than that of SARS-CoV RBD.

Therefore, recombinant SARS- CoV-2 RBD and SARS-CoV RBD antibodies can be used as SARS-CoV-2 virus adsorption inhibitors.4 It has been reported that CR-3022, a monoclonal antibody acting on SARS-CoV, can bind to the RBD non-ACE2 binding site of SARS-CoV-2, which may inhibit the binding of RBD of SARS-CoV-2 and ACE2.

Therefore, CR3022 can be used alone or in combination with other drugs to inhibit the identification between SARS-CoV-2 RBD and ACE2. However, some other definite neutralizing antibodies of SARS-CoV RBD, like m396 and CR3014, fail to bind to SARS-CoV-2, though they contain domains aiming at the ACE2 binding site.

This interesting phenomenon implies that the difference between the RBD of SARS-CoV-2 and the RBD of SARS-CoV is a potential target to develop novel monoclonal antibodies of SARS-CoV-2.5 A latest study reported that REGN-COV2, a cocktail of two potent neutralizing antibodies (REGN10987 + REGN10933) which targets nonoverlapping epitopes on SARS-CoV-2 spike cell fusion inhibitor targeting the S protein−ACE2 interaction to prevent viral entry into host cells.10 Interestingly, two independent research groups have found that Withaferin A (WFA), a steroidal lactone with anti-inflammatory and antitumorigenic properties, may bind to SARS-CoV-2 spike protein so that it may interfere with the virus entry into cells.11

Also, it is reported that griffithsin can inhibit the SARS-CoV from entering host cells via binding to spike glycoprotein, but the effect of griffithsin on SARS-CoV-2 remains unknown.12

CD147

CD147, also known as basigin or EMMPRIN, is one of the highly glycerogelatin transmembrane proteins of the immuno- globulin superfamily, and it also acts as a main upstream stimulator of matrix metalloproteinases (MMPs).

Recently, it is demonstrated to mediate the entry of SARS-CoV-2 into host cells as well as ACE2, since the spike protein of SARS-CoV-2 can bind to not only ACE2 but also CD147.13 As the expressions of the CD147 and MMPs in inflammatory reactions are usually high, inhibiting CD147 may have a positive effect on the severe acute respiratory syndrome triggered by SARS-CoV-2.14 Therefore, CD147 may be a potential target against SARS-CoV-2.

Recently, there is a clinical trial entitled “Clinical Study of anti-CD147 Humanized Meplazumab for Injection to Treat With 2019 nCoV Pneumonia” (ClinicalTrials.gov Identifier: NCT04275245) underway in phase II in China. This trial aims to inhibit CD147 from binding to the spike protein via monoclonal antibodies and then inhibit the subsequent infection.

Besides, studies have demonstrated that azithromycin could decrease the expression and action of MMP,15 but it remains unknown whether azithromycin directly changes the expression levels of CD147 receptor. Interestingly, it has been reported that doxycycline, an antibiotic analogue of tetracycline, can reduce the level of CD147 in patients with chronic periodontitis16 or a gallbladder carcinoma cell line,17 but its role in COVID-19 needs more research.

ACE2

Angiotensin-converting enzyme-related carboxypeptidase (ACE2) is a novel enzyme that was found in 2000. ACE2 mainly has 3 functions in humans: first. It can hydrolyze the carboxy-terminal leucine of angiotensin into generating angiotensin 1−9, and then the angiotensin can be hydrolyzed in smaller angiotensin peptides via cardiomyopathy in vivo or via ACE in vitro.

Interestingly, ACE2 mainly expresses in the kidney and heart.18 As ACE2 has organ-specific and cell- specific expression, it plays an important part in the renin- angiotensin system of the kidney and heart. Second, it has been observed that ACE2 expresses in the lung AT2 alveolar epithelial cells, which are particularly prone to viral infection.

PENETRATION

Furin

Furin, also known as paired basic amino acid cleaving enzyme (PACE), has substrate specificity to carve up specific amino acid sequence.42 Furin mediates the entry of SARS-CoV-2, but not SARS-CoV.

Furin can preactivate the spike protein of SARS-CoV-2 and, change the conception of the spike protein to expose the binding and fusion domains and then promote the TMPRSS2-mediated entry of the SARS-CoV-2 into the host cells.43 Because of furin, SARS-CoV-2 has a higher infection rate, and some clinical patients to COVID-19 with severity may refer to it.

A recent study has certified that furin carves up the spike protein efficiently and increases the affinity between spike protein and ACE2, which leads to higher pathogenicity of the SARS-CoV-2.43 Besides, although it is established that furin is generally membrane-bound, a study has demonstrated that there is also a secreted form of furin, which may promisingly facilitate the cleavage of the spike protein of SARS-CoV-2 around the host cells.44

Besides, furin has been detected in the T-cells which are activated when the body is infected by the virus. This may lead to a feedforward loop of furin-mediated viral replication and result in a hypersensitive immunological response (cytokine storm) in some patients, which can cause lethal multiorgan failure.45

These studies suggest that furin may be a potential antiviral target. The recent drugs inhibiting the activity of furin are furin Inhibitor I, furin convertase inhibitor (Chloromethyl ketone), and peptidyl-chloromethyl ketones.46 Besides, a variant of naturally occurring serine protease inhibitor called α-1 antitrypsin Portland (α1-PDX) can inhibit furin.47 These drugs have been applied to HIV infection, so they are relatively safe.

Besides, it has been reported that a nonpeptide 2,5- dideoxycyclic vitamin small molecule inhibitor could bind to furin with two sites, and this small molecule inhibitor can be considered as a potential compound against SARS-CoV-2.

Moreover, scientists have designed a 2,5-dideoxy streptamine- derived inhibitor based on the crystal structure of furin, and this compound can form a complex with furin, which can inhibit the activity of it.48 It is worth noting that furin is required for some normal developments and cellular processes, such as the regulation of blood clotting, tumor progression, and growth signaling.49

Therefore, inhibiting furin activity for a long time may bring unpredictable risks, so brief therapy targeting furin may lead to good results in the treatment. These small molecule inhibitors and other promising orally active agents also need tests to assess their antiviral effect against SARS-CoV-2.

TMPRSS2

Coronaviruses penetrate host cells mainly via fusion. In addition to the spike protein binding to ACE2, its fusion requires cellular proteases to proteolysis the spike protein, such as cathepsins, cell surface transmembrane protease/serine 2(TMPRSS2) proteases, and trypsin.50 TMPRSS2 is one of the proteases mediating SARS-CoV-2 into the host cells.

It can be prime not only in the cleavage of S2 but also in the cleavage of ACE2, which is required for the initiation of membrane fusion as well. The expression profile of TMPRSS2 is extensive, while the membrane fusion domain is conservative.

Research has been reported that camostat mesylate, the serine protease inhibitor can impact the activity of TMPRSS2, and efficiently inhibit the virus from entering the TMPRSS2+ cells instead of the TMPRSS2− cells, while another research study reported that there is no death and other serious incidents that occur after knocking out TMPRESS2 in mice,43 so TMPRESS2 can be considered as an effective therapeutic target for COVID-19.

It is noteworthy that serine protease inhibitor camostat mesylate has been applied in the treatment of pancreatic inflammation disease in Japan for safety, so it can also be regarded as a promising drug for COVID-19.51 A recent research study in Japan has reported that nafamostat mesylate, a comparable drug of camostat mesylate, can inhibit the membrane fusion triggered by TMPRESS2 at a lesser concentration than camostat mesylate.

This discovery indicates that nafamostat mesylate can be deemed as a potential agent for combating SARS-CoV-2. As these two drugs have been used in Japan for several years, toxicity and adverse reactions of them are known, so they can be used for COVID-19 without too much safety worry.52

Another potential inhibitor of TMPRSS2, bromhexine hydrochloride, which is an FDA approved agent used to expectorate and relieve cough can also be considered, although it requires clinical trials to assess its effect on the SARS-CoV-2.53 Research studies on TMPRSS2 with other viruses are also worth referring.

For example, a study has found that some Benzo seleno xanthene analogues can down-regulate the expression of the TMPRSS2 gene via stabilizing the TMPRSS2 G-quadruplex structure, which is quite important for gene transcriptional activity of TMPRSS2 to inhibit influenza A virus propagation in vitro.54

The studies above suggest that these Benzo seleno xanthene analogues may also be potential candidates against SARS-CoV- 2.

Also, a study has been noted that nelfinavir may inhibit cellular proteases that are required for SARS-CoV-2 fusion activation, including TMPRSS2.55 Meanwhile, studies have shown that Cbz (Carboxybenzyl)-Phosphoono-LYS(OPh)2 irreversibly inhibits TMPRSS2 activity, so that it may be a potential drug against SARS-CoV-2.56

Moreover, molecular docking study is designed to find drugs suppressing TMPRSS2, and a recent study identified four potential inhibitors against TMPRSS2: drugs Rubitecan and Loprazolam, and compounds ZINC000015988935 and ZINC000103558522. Nevertheless, in vitro and in vivo experiments remain to prove their effectiveness against SARS-CoV-2.

CONCLUSION

In this paper, the related targets and possible drugs in each replication cycle of the virus are reviewed, which provides a basis for further development of SARS-CoV-2 drugs. For the relatively conservative proteins of SARS-CoV-2, such as entry- related protein, fusion-related protein, and RdRp, research studies mainly originate from homologous targets of other viruses.

The drugs about Mpro or PLpro can be searched via large data, such as screening for repurposed drugs and related compounds. After that, in vitro and in vivo experiments should be conducted in time to evaluate the clinical effects of COVID-19. Besides, there are not enough studies on the process of SARS-CoV-2 assembly and release, suggesting that it may be a direction for finding SARS-CoV-2 drugs. Favipiravir