Current Potential Therapeutic Approaches against SARS-CoV-2: A Review

The ongoing SARS-CoV-2 pandemic is a serious threat to public health worldwide and, to date, no effective treatment is available. Thus, we herein review the pharmaceutical approaches to SARS-CoV-2 infection treatment. Numerous candidate medicines that can prevent SARS-CoV-2 infection and replication have been proposed. These medicines include inhibitors of serine protease TMPRSS2 and angiotensin converting enzyme 2 (ACE2). The S protein of SARS-CoV-2 binds to the receptor in host cells. ACE2 inhibitors block TMPRSS2 and S protein priming, thus preventing SARS-CoV-2 entry to host cells. Moreover, antiviral medicines (including the nucleotide analogue remdesivir, the HIV protease inhibitors lopinavir and ritonavir, and wide-spectrum antiviral antibiotics arbidol and favipiravir) have been shown to reduce the dissemination of SARS-CoV-2 as well as morbidity and mortality associated with COVID-19.


Introduction
Coronaviruses contain positive-sense, single-stranded RNA, with a genome size ranging from 26-32 kb, and five structural proteins, and are classified into four categories: alpha, beta, gamma, and delta [1,2]. Human coronaviruses are alpha and beta coronaviruses which can cause respiratory and gastrointestinal tract infections [2]. The severe acute respiratory syndrome (SARS) outbreak between November 2002 and July 2003 (nine months) resulted in more than 8000 total cases and 774 deaths, with a fatality rate of 9.6% [3]. Middle East respiratory syndrome (MERS) was reported in 2012 resulting in more than 2400 cases and 858 deaths, with a fatality rate of 34.4%. Subsequently, in late December 2019, an unspecified case of pneumonia was reported in Wuhan, Hubei Province, the People's Republic of China [1][2][3]. COVID-19 is the official name given by the WHO to the disease caused by SARS-CoV-2 infection. It has since been observed that the virus could spread from human to human [4]. Its incubation period is 2 to 14 days with various clinical presentations: asymptomatic, mild to severe illness, and mortality [5]. Symptoms include fever, cough, difficulty breathing, malaise and fatigue, gastrointestinal symptoms (decreased appetite, vomiting, watery diarrhea, and dehydration), loss of taste and smell, sore throat, rhinorrhoea, severe pneumonia, and acute respiratory distress, which can lead to multiple organ failure and death. The SARS-CoV-2 virus is mainly spread via airborne/aerosol particles; the virus has been observed to remain viable and infective for over 3 h in the air [6,7]. SARS-CoV-2 infection is a highly communicable disease, and this pandemic has been designated a world public health emergency by the World Health Organization (WHO) [7]. However, SARS-CoV-2 has many potential natural, intermediate, and final hosts, as do other viruses; thus, major problems in the prevention and diagnosis of viral infection are raised [8]. In this paper we discuss the genetic structure of SARS-CoV-2 and its mechanism of pathogenesis. We include consideration of the phylogenetic analysis of the SARS-CoV-2 genome, multiple sequence alignment analysis, and therapeutic approaches to SAR-Co-V-2 infection.

SARS-CoV-2 Genetic Structure and Pathogenic Mechanism
The SARS-CoV-2 genome codes for more than 20 distinct proteins. At least four structural proteins are present in coronaviruses, namely spike (S), envelope (E), membrane (M), and nucleocapsid (N) proteins ( Figure 1). S proteins, which are involved in host attachment and virus-cell membrane fusion, determine the host range for viral infection ( Figure 2) [9].   The SARS-CoV-2 main protease (Mpro) is recognised as one of the most essential viral proteins. SARS-CoV-2 Mpro is more than 96% similar to SARS-CoV Mpro. During viral translation, SARS-CoV-2 Mpro cleaves 11 polyproteins to polypeptides that are required for transcription and replication [10]. Some of the candidate drugs that can prevent SARS-CoV-2 viral replication target Mpro, such as remdesivir, griffithsin, nafamostat, disulfiram, lopinavir/ritonavir, nelfinavir, danoprevir and favipiravir [11].

Phylogenetic Analysis of SARS-CoV-2 Genome
A sequence alignment and phylogenetic analysis of SARS-CoV-2 genome is shown in Figure 3. The phylogenetic tree is primarily divided into three clades [12]. Clade I consist of SARS-CoV and Bat-SL-CoV genomes which share a sequence identity ranging from 88% to 99%. Clade II consist of 13 complete genomes of coronavirus and MERS-CoV genomes which share a sequence identity from 78% to 89%. Clade III consist of 23 SARS-CoV-2 and Bat-SL-CoV complete genomes which share a sequence identity ranging from 89% to 100%; the SARS-CoV-2 genomes isolated from human samples show a sequence identity ranging from 98% to 100% [13]. A particularly interesting observation from the analysis was that there is no major divergence in the SARS-CoV-2 genome sequence of different SARS-CoV-2 virus genomes isolated from different countries, as shown in Figure 3. The sequence alignment of the SARS-CoV-1 (Bat, PDB ID: 3TNT) and the SARS-CoV-2 (human, PDB ID: 7MBI) main proteases reveals that the amino acid sequence is conserved with a sequence identity of 96%; differences between these genomes are shown in Figure 4 at specific positions [13,14].  Multiple sequence alignment analysis of the amino acid sequence of SARS-CoV-1 and SARS-CoV-2 Mpro. Amino acids marked underneath with an arrow represent catalytic residues; residues marked underneath with * represent substratebinding residues of various subsites.

Therapeutic Approaches to SAR-COV-2 Infection
To identify therapeutic agents that are effective against SARS-CoV-2 infection, extensive research on the structure and pathogenesis of COVID-19 is in progress [15]. Therapeutic approaches to COVID-19 can be categorized into virus-based therapy and host-based therapy, as shown in Figure 5.

Virus-Based Therapy
Viral nucleic acids consist of nucleosides and nucleotides. Drugs capable of attacking nucleotides, nucleosides, or viral nucleic acids can affect the activity of a broad range of coronaviruses and other viruses, as shown in Table 1 [16]. Possible targets for antiviral therapy include major enzymes and proteins involved in SARS-CoV-2 viral replication. The PLpro enzymes and papain-like protease of SARS-CoV and MERS-CoV have been shown to exert proteolytic, deubiquitylating, and deISGylating activities. Studies have shown that lopinavir-ritonavir is the most potent protease inhibitor as shown in Table 1 [17]. The main SARS-CoV-2 immunogen antigen is the Spike glycoprotein with membrane anchor, which plays an important role in the interaction between host cells and viruses. Studies have shown that certain monoclonal antibodies can target the receptor binding domain (RBD) subunit epitopes and inhibit viral cell receptor binding, whereas other monoclonal antibodies bind to the S2 subunit and disrupt viral cell fusion [18]. A study using the CR3022 neutralising antibody of SARS-CoV shown in Table 1 [19]. Earlier trials also showed that adoptive transfer of plasma containing anti-MERS-COV-S antibodies had the ability to prevent infection and accelerate viral clearance.

Host-Based Therapy
Viral entry of SARS-CoV-2 depends on the priming of its spike protein and on transmembrane protease 2 (TMPRSS2). Further studies have shown that camostat mesylate, a serine protease inhibitor, can block TMPRSS2 activity and is thus considered as a therapeutic candidate as shown in Table 2 [33]. Other research indicates a pH-and receptor-dependent endocytosis when coronavirus is introduced into the host cell. AP-2-associated protein kinase 1 (AAK1), a host kinase, controls clathrin-mediated endocytosis [34]. Since the virus structure is now established, various inhibitors have been tested in cell-based systems for their ability to prevent viral entry and replication within the host body, as shown in Table 2 [35]. These include spike (S) protein inhibitors, S-cleavage inhibitors, helicase and protease inhibitors, fusion core blockers, HCB monoclonal antibodies, RBD-ACE2 blockers, antiviral peptides, siRNAs, and antifreeze eutralizati antibodies [35,36]. The following section concentrates on the possible therapeutic treatment options based on our limited knowledge of SARS-CoV-2.

Neutralizing Antibodies
In general, coronavirus infection begins with the entry of the viral S protein, which binds to the cell surface. This S protein fuses with the cell membrane and facilitates the syncytial development and transmission of viral nucleocapsids into the cell for further replication [35]. Studies have shown that neutralization of the S protein RBD of SARS-CoV [36] and MERS-CoV [38][39][40] by antibodies can be effective against these diseases. Neutralisation of antigens can be highly useful in COVID-19 treatment, given that the S protein RBD sequence of SARS-CoV-2 is similar to those of SARS-CoV and MERS-CoV [37]. Critical COVID-19 patients are currently treated with immunoglobulin G [35,36]. FcR plays a role in inflammation in the lung; therefore, inflammation in COVID-19 can be reduced by blocking FcR activation. Thus, intravenous administration of immunoglobulins can be effective in the treatment of pulmonary inflammation, as shown in Table 3 and Figure 6 [41].

Antiviral Proteases
Lopinavir has been suggested as a treatment choice for persistent COVID-19 infection [41], as it inhibits the activity of coronavirus proteases. In addition, an inhibitor of vinyl sulfone protease has recently been proposed for treating patients with COVID-19 and can potentially be used widely as an anti-coronaviral agent [42]. Recently, the elucidation of the SARS-CoV-2 Mpro structure has brought about a breakthrough in antiviral studies, and numerous new drug candidates have been developed since. The Deep Docking platform, a virtual screening platform for structural proteins, containing approximately 1.2 billion molecules and 1000 suspected SARS-CoV-2 Mpro proteins, was recently established [42].
Traditional Indian medicine, which includes Ayurveda, Siddha, Unani and Yoga, naturopathy, and homeopathy, is one of the oldest therapies in human history and has been used to treat various diseases [46]. However, numerous other drugs for COVID-19 patients are still being tested. Teicoplanin is a staphylococcus glycopeptide [45] recommended for the treatment of complex COVID-19 cases and S. aureus infections [45], and has been shown to be effective in treating complications in these patients. In a clinical trial, danoprevir, a viral protease inhibitor, led to substantial improvement in patients with COVID-19 [47]. A summary of the development of therapeutics against COVID-19 is shown in Figure 5.

SARS-CoV-2 Cell Entry Inhibitors Inhibitors of TMPRSS2 Serine Protease
Transmembrane protease serine subfamily 2 (TMPRSS2), an airway and alveolar cell serine protease, is regulated by the cleavage and activation of the SARS-CoV spike protein (S protein), which is necessary for membrane fusion and host cell entry [48]. Camostat mesylate is a clinically proven serine defence inhibitor that partially prevents infection of HeLa cells expressing the ACE2 and TMPRSS2 by HCoV-NL63 and SARS-CoV-2 [49].

Nafamostat Mesylate
Nafamostat mesylate (FUT-175; CAS number: 81525-10-2) is an artificial serine protease inhibitor clinically approved in Japan for the treatment of acute pancreatitis, intravascular coagulation dissemination, and extracorporeal circulation antioxidation [49,50]. Through screening of approximately 1100 drugs for those that can inhibit the fusion of TMPRSS2, the FDA determined that nafamostat mesylate inhibited the fusion of TMPRSS2expressing Calu-3 host cells with the MERS-CoV S protein [49,50].

Angiotensin Transforming Enzyme 2 (ACE2) Inhibitors and Antimalarial Drugs
SARS-CoV and corresponding coronaviruses interact directly with the host cell exoprotease and metallocarboxypeptidase (ACE2, S) via their S proteins, with angiotensin converting enzyme 2 (ACE2) catalysing the conversion of angiotensin I into nonapeptide angiotensin and angiotensin II into angiotensin 1-7 [53,54]. Unfortunately, the ACE inhibitors commonly used against hypertension and chronic heart failure cannot inhibit ACE2 [53,54]; however, other drugs and compounds have been shown to inhibit ACE2 [53].

Chloroquine Phosphate and Hydroxychloroquine
Chloroquine has been used for decades in malaria prophylaxis and for the treatment of chronic Q fever and autoimmune illnesses [57]. Hydroxychloroquine is transformed into chloroquine in vivo. These drugs have recently been shown as possible broad-spectrum antiviral medications [58]. Chloroquine phosphate inhibits ACE2 terminal phosphorylation and increases pH, both of which constitute the antiviral mechanisms of chloroquine and hydroxychloroquine [57,58].

Inhibitors of the Replication, Membrane Fusion, and Assembly of SARS-CoV-2 Lopinavir/Ritonavir (Kaletra™)
Lopinavir (ABT-378) was developed in 1998 to overcome HIV resistance to the protease inhibitor ritonavir (ABT-538), which is caused by a valine mutation at position 82 (Val 82) on an active site of HIV protease in response to ritonavir treatment [38]. As the metabolism of lopinavir is strongly inhibited by ritonavir, lopinavir has been administered at oral dosages exceeding ritonavir by >50-fold in rat, dog, and monkey-plasma for an in vitro EC50 for lopinavir after 8 h [39]. Lopinavir-ritonavir treatment was associated with inconsistencies in clinical improvement, but showed comparable mortality with the standard-care treatment after 28 days of treatment [38,39].  [59]. Umifenovir prevents viral host cell entry by inhibiting fusion of the viral envelope and host cell cytoplasmic membrane through inhibition of clathrin-mediated endocytosis, thereby preventing virus infection [60].
Inhibitors of SARS-CoV-2 3CLpro Protease 3CLpro (also known as Mpro) is the major betacoronavirus protease necessary for viral RNA translation in polyprotein synthesis [10,30]. Recently, the x-ray structures and α-ketoamide complex of SARS-CoV-2 3CLpro have been reported [10,30,61]. Two compound pulmonary tropism pyridine-containing α-ketoamides, called 13a and 13b, exerted pharmacokinetic properties at sufficient concentration in the lungs and bronchialalveolar liquid lavage of mice within 4 to 24 h of administration [47]. N3, formerly known as Michael acceptor inhibitor, was developed using a computer-aided method. N3 can specifically inhibit the Mpro of multiple coronaviruses, including SARS-CoV and MERS-CoV, and has shown potent antiviral activity against infectious bronchitis virus in an animal model [51,52,61]. N3 was shown to form a covalent bond with SARS-CoV-2 3CLpro, thereby acting as an irreversible inhibitor of SARS-CoV and MERS-CoV 3CLpro [51,52].

SARS-CoV-2 Mpro Inhibitor
The SARS-CoV-2 Mpro structure in the apo form and α-ketoamide bound with the protein form a dimer crystallographic composed of two monomers of identical conformations [62]. Each protomer is made up of three domains, as shown in Figure 7A,B, Domain I (residues 8-101) and II (residues 102-184) interface to form the active site of the protein, where α-ketoamide derivative bound complex and composed of a Cys145-His41 dyad with an antiparallel β-barrel structure. Domain III (residues 201-303) contains five α-helices, connected to domain II by a long loop region (residues 185-200), with the substrate-binding site located in a cleft between domain I and domain II. It has shown several highly conserved interactions and forms a catalytic bond with the His41 and Cys145 and adjacent residues in the substrate binding cleft such as Gly143 and Ser144 dyad. SARS-CoV uses a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro) to process and cleave its long polyprotein precursor into individually functional non-structural proteins [63,64]. In the active site region of SARS-CoV-2 Mpro, the S1 residues contributed by Cys145, Gly143, and Ser144 also serve as the oxyanion hole. Bulky Gln189 and Pro168 residues form the S4 site, the S1 residue is His163, while Glu166 and Gln189 are located at the S2 position ( Figure 8A). The Mpro recognizes and binds specific residues at each subsite of the peptide substrate to determine the initiation of proteolysis and the production of non-structural proteins for the formation of the replication-transcription complex. Crystal structures of Mpro have revealed that the most variable regions are the helical domain III and surface loops, and that the substrate-binding pocket (located in a cleft between domain I and domain II) is highly conserved among Mpro in all coronaviruses; this suggests that antiviral inhibitors targeting this pocket should have wide-spectrum activity against coronaviruses, as shown in Figure 8B. A previous study proposed that Mpro has a substrate-recognition pocket that is highly conserved among all coronaviruses and that this pocket could serve as a drug target for the design of broad-spectrum inhibitors. The recent discovery of new coronaviruses, and the accumulation of structural data for Mpro from coronaviruses of various species, has provided the opportunity to further examine this hypothesis.

Nanomaterials Approach for Anti-Coronavirus
Nanomaterials, such as silver colloid, titanium dioxide, and diphyllin nanoparticles, are considered promising antiviral agents and drug-delivery platforms for the effective management of coronavirus infection (Tables 4-6) [65][66][67][68][69][70][71][72]. Small interfering RNAs (siRNAs) are highly efficient in reducing the replication of RNA viruses, such as coronaviruses. The efficacy of siRNA-based treatments strictly depends on specific targeting of the viral sequence of interest and targeted cellular delivery of therapeutic siRNA [73]. In this context, nontoxic, biocompatible nanocarriers composed of polymers, lipids, polymer/lipid hybrid nanoparticles, nanohydrogels, silica, dendrimers, iron oxide nanoparticles, or gold nanoparticles are considered as promising siRNA-delivery platforms [74]. These nanocarriers can improve siRNA stability by preventing enzymatic degradation. For inhalable antiviral siRNA loading and aerosol-based delivery of antiviral siRNA in the lungs, polymer/lipid nanocarriers have shown promising outcomes [73,74].
Antiviral nanoparticles have been used as potential immunostimulatory agents for vaccine development. For example, gold nanoparticles conjugated with swine transmissible gastroenteritis virus have been used to activate macrophages, induce interferon production, and increase anti-coronavirus neutralizing antibody levels in vaccinated animals [75]. Similarly, conjugates of ribonucleic acid and ferritin-based nanoparticles have been used as molecular chaperons to develop a vaccine against MERS-CoV [74,75]. The vaccine has been shown to induce a strong T cell response and promote interferon production. Currently, nanotechnology is playing an increasingly important role in antiviral therapy for coronaviruses [73,75]. Nanomaterials have been developed specifically to improve the delivery of biotherapeutics across physiological barriers [73][74][75]. A broad range of potential nanodevices, such as nanosensors, nano-based vaccines, and smart nanomedicines, offer great hope for combating current and future mutated versions of coronaviruses [72].

Conclusions
As vaccines will not be available for the general population until the end of 2020, approved off-label and experimental drugs that are effective against COVID-19 have been identified, including TMPRSS2 and ACE2 inhibitors, antifungal and antimalarial products, antiviral medicines inhibiting viral RdRp, proteases, virus/host cell membrane fusion inhibitors, and antiviral phytochemicals. In addition to infection preventive strategies that have been implemented in many countries, such as quarantine of infected persons, mobile monitoring of contacts, protection of persons at high risk of infection, restriction of national curfew, and urgent vaccine production and supply, trials of these drugs have been conducted by the WHO and the European Union. This review of therapeutic approaches to COVID-19 aims to facilitate the production of drugs and vaccines against this disease. In future studies, furin inhibitors may be investigated as a strategy for developing SARS-CoV-2 vaccines.

Conflicts of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.