key: cord-0777515-tuclasjf
authors: Rajarshi, Keshav; Khan, Rajni; Singh, Mrityunjay K; Ranjan, Tushar; Ray, Sandipan; Ray, Shashikant
title: Essential functional molecules associated with SARS-CoV-2 infection: Potential therapeutic targets for COVID-19
date: 2020-11-18
journal: Gene
DOI: 10.1016/j.gene.2020.145313
sha: 79cb6e571bd57e9c429a032b485273f5f21ae0d9
doc_id: 777515
cord_uid: tuclasjf

The whole world is still suffering substantially from the coronavirus disease 2019 (COVID-19) outbreak. Several protein-based molecules that are associated with the Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which are essential for its functionality, survival, and pathogenesis have been identified and are considered as potential therapeutic targets. These protein-based molecules are either structural/non-structural components of SARS-CoV-2 or host factors, which play a crucial role in this infection. Developing drug molecules against these essential functional molecules to hinder their regular functioning and associated physiological pathways could be promising for successful clinical management of this novel coronavirus infection. The review aims to highlight the functional molecules that play crucial roles in SARS-CoV-2 pathogenesis. We have emphasized how these potential druggable targets could be beneficial in tackling the COVID-19 crisis.

The 21 st century has witnessed severe pneumonic pandemics caused by various coronaviruses (CoVs) . In 2003, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) broke out, and the incidence of respiratory ailments caused by the virus with a lethal rate of approximately 10% was reported from five different countries [1, 2] . During the year 2012, the Middle East Respiratory Coronavirus (MERS-CoV) caused a similar pneumonic outbreak in the Arabian Peninsula. The virus had a case fatality rate (CFR) of approximately 35% [3, 4] . More recently, a new coronavirus emerged at the end of the year 2019, in December in the city of Wuhan, situated in the Hubei province of China. This novel coronavirus was named SARS-CoV-2 or Severe Acute Respiratory Syndrome Coronavirus-2 [5] . This novel coronavirus was encountered for the first time and was reported to cause respiratory disorders in infected individuals.

After the outbreak in China, SARS-CoV-2 infections are promptly reported in countries like the USA, India, Brazil, Russia, Colombia, Peru, Spain, Mexico, Argentina, etc. [6] . At the beginning of the outbreak, the symptoms of the novel coronavirus infection were found to be somewhat similar to that of the regular flu, caused by the seasonal influenza virus, and hence were confused with the same disease [7] . But as soon as the situation got 

Spike (S) proteins are class I fusion proteins expressed on the viral surface. These proteins are densely glycosylated and consist of a large ectodomain, i.e., a single-pass transmembrane domain which provides anchorage for the proteins to the lipid bilayer as well as to a small intracellular segment. S1 and S2 are the two subunits comprised of the ectodomain, which forms homotrimers. The S1 subunit consists of a C-terminal functional domain that is involved in binding with the receptor. The S2 subunit comprises a cytoplasmic domain that assists in the fusion of viral envelope with the membrane of the host cell through the endosomal pathway, a transmembrane domain, and a fusion peptide called heptad repeat 1 and 2 (HR1 and HR2) [39] . The S protein is present in a pre-fusion form on the surface of a virus particle [40] . After the contact of the virus with the host cell, the host cell membrane proteases like transmembrane protease serine-2 (TMPRSS2) are responsible for the priming of S protein, to carry out internalization efficiently via the process membrane wrapping [41, 42] . The structural elucidation of SARS-CoV-2's receptor-binding domain (RBD) reported that its binding affinity with the ACE2 receptor is approximately ten times stronger than the previously encountered SARS-CoV. Also, the S2 domain of the SARS-CoV-2 was reported to be relatively more flexible than the SARS-CoV [43]. Another major difference between the structure of spike proteins of SARS-CoV and SARS-CoV-2 is the position of RBDs in their respective down conformations. In the case of SARS-CoV, the RBD packs tightly against the NTD (N-terminal domain) of the neighbouring protomer, in the down protomer, whereas in the case of SARS-CoV-2, RBD is angled closer to the trimer's central cavity in the down conformation [44] . The spike protein of SARS-CoV-2 bears proteolytic sites known as the S1/S furin-like rift spot, which is not present in SARS-CoV and is reported to enhance the pathogenicity of the virus, thus distinguishing both the viruses. It has also been reported that the furin-like rift spot also results in the enhancement of the tissue tropism of the viruses [45, 46] . The spike protein is exposed to an immense evolutionary pressure as it is the first contact site between viruses and the host cells. The transmission and infectivity of the viruses are greatly influenced by the changes in the spike protein. In the case of the SARS-CoV-2, the spike protein underwent several changes like a furin-like cleavage site, and changes at the binding sites of the receptors are being considered as the reason behind species jumping and transmission among humans efficiently. Additionally, it has also been reported that the SARS-CoV-2 forms syncytium, which allows the spreading of viruses via cell-cell fusion and might also contribute towards its rapid infectivity [47] .

The 3C-like protease, encoded by Nsp5, is also known as the main protease (M pro ). The M pro is known to be the first protein to get auto-cleaved and then further leads to the cleavage of polyprotein into discrete members of NSPs at the LeuGln↓ (Ser, Ala, Gly) cleavage site. The stable active form of the M pro is achieved in the form of an octamer whilst its dimer and monomer subsist in equilibrium [48] . The structural arrangement of SARS-CoV-2 is nearly 96% similar to that of the SARS-CoV. It was revealed from the SARS-CoV-2 M pro crystal structure that the non-polar amino acids in the interface of dimer replaced the polar ones. This mutation led to the enhanced catalytic activity of the dimer, whereas the dimer dissociation constants were reported to have remained the same [49] . For the replication and production of virus proteome, protease is an essential requirement, and hence, M pro is suggested as one of the potential targets for curing COVID-19.

Very complex protein machinery is involved in the viral genome replication, as well as the transcription of SARS-CoV-2. The proteins associated with this complex are part of a large polypeptide chain and are translated from ORF1a and ORF1b. On the whole, the SARS-CoV-2's RNA dependent RNA polymerase (RdRp) complex structure corresponds to that of the SARS-CoV. The RNA dependent RNA polymerase, i.e., Nsp 12, is the key component of the enzyme complex. The complex formation is assisted by other co-factors like Nsp7, Nsp8, along with Nsp12 and the development of viral genomic RNA is catalyzed. The templatedirected RNA synthesis is catalyzed at the central grove of the complex. The positively charged nascent RNA strands exit path, which is also accessible to the solvents, facilitates the electrostatic interaction between the enzyme complex and nascent RNA strand [50, 51] .

The Nsp12 (RNA polymerase unit) can be considered as a reassuring potential target for the development of therapeutic drugs like remedesivir (CID: 121304016) and viral inhibitors.

Drugs like remedesivir inhibit the RdRp activities and have now entered into clinical trials for developing a promising cure for COVID-19.

The transmission of SARS-CoV-2 usually occurs via close contact among individuals and the droplet infection, as well as via stool, sweat, exposure to respiratory secretions, and urine [52] [53] [54] . Upon the entry inside the body, the virus binds to pneumocytes and enterocytes, which are the primary target cells, as a result of which, the virus instigates an infection and replication cycle. CoVs like SARS-CoV-2 may also target other cells, including the kidney's tubular epithelial cells, neuronal cells, renal epithelial tubules, and also cerebral cells and immune cells [52, 53] . [58] . Ultimately, by forming a structure that resembles a bud, the viral particle buds off and is shot out of the ERGIC complex [59] . At last, vesicle formation is carried out by the mature virions. These virus particles are thus released outside into the extracellular region by fusion of the vesicles, formed by virions, with the plasma membrane [59, 60] . The mechanism of pathogenesis, as well as the regeneration and life cycle of SARS-CoV-2, is described in Figure 1 .

The coronavirus infection usually induces a surge of pro-inflammatory chemokines and cytokines, thus resulting in functional deterioration and damages to the tissues of infected organs, especially lungs, and even leads to failure of lung and deaths in several cases [61] . Intriguingly, the C-terminus region of S-protein showed more conserved amino acids compared to N-terminus ( Figure 2A ). On the other hand, analysis of M-protein from different strains (reported from different regions/country) fetched conserved motifs throughout the polypeptide chain ( Figure 2B ) with comparatively less variation at the N-terminus. We are speculating that these motifs probably have a direct role in the viral maturation process, and therefore, targeting these highly conserved regions may open some promising avenues in drug discovery against this novel respiratory pathogen.

The spike (S) protein of SARS-CoV-2 is expressed in a trimeric form on the surface of the virus and imparts the crown-like appearance (hence the name coronavirus), which is the prime structural protein. The host-virus interaction, as well as the determination of tissue tropism or host tropism, is mediated by the spike protein as the attachment of spike protein with the receptors present on the host cell facilitate the invasion of the virus [62] . TMPRSS2, which is a host cell protease, cleaves the spike protein into two subunits, i.e., S1 and S2. S1

primarily binds with the surface receptor expressed on the host cell, whereas the virus to cell and cell to cell membrane fusion is carried out by the S2 subunit of the spike protein.

Activation by cleavage and structural integrity of the spike protein plays a significant role in the virulence of CoVs and their invasion [63] . Spike protein or the host cell receptors can be 

In the case of CoVs, NSPs are engaged as a prominent functional protein in transcribing RNA, translating, and synthesizing proteins. Processes like the synthesis of proteins, RNA translation, transcription, modification, processing, host infection, and viral replication are all carried out by NSPs. The functions of the major NSPs are listed in Table 1 . The biological functions of NSPs like RNA dependent RNA polymerase, PLpro, helicase, and 3CLpro are well known, and due to the clarity about their essential active site for the enzyme, they are considered as major targets for developing small-molecular inhibitors. Nsp12-RdRp has been given significant importance in several studies conducted to find out inhibitors for CoVs.

Nsp12-RdRp can be considered as a potential drug target since its targeted inhibition could not induce any significant side effects and notable toxicity on host cells [65] .

Nsp12 is a conserved coronavirus protein which acts as an RNA-dependent RNA polymerase and is a crucial component of the viral transcription and replication complex. The polymerase's RdRp domain consists of a conserved Ser-Asp-Asp sequence motif and is situated at the C-terminus [66] . A primer of length up to six nucleotides can be de novo synthesized by the aid of Nsp8, which can be employed in RNA synthesis using RdRp.

Additionally, the enhancement in Nsp12's RdRp activity along with an increase in the binding of Nsp12 to the RNA is facilitated by the Nsp7-Nsp8 complex [67] . The results of the virtual ligand screening study suggested that some anti-bacterial drugs like novobiocin 

The release of Nsp1, Nsp2, and Nsp3 upon the cleavage of replicase polyprotein's Nterminus is facilitated by PLpro. The released NSPs, i.e., Nsp1, Nsp2, and Nsp3, are crucially 

Nsp13, also known as helicase, which includes a Hel (helicase) domain along with an Nterminal MBD metal-binding domain (MBD), is a protein with multiple functionalities. A total of 26 cysteine residues constitute the N-terminal structures and form a Zn 2+ binding domain, whereas the conserved sequence motif at the C-terminus constitutes the Hel domain.

Unpacking or unwinding of the double-stranded DNA/RNA is carried out by Nsp13 in an NTP-dependent manner along 5´ to 3´ direction [72] . Notably, the SARS-Nsp13 sequence has been reported to be a vital component for CoV replication and has also been reported to be conserved, which makes it a potential target for developing antiviral drugs. Very few studies and reports have discussed the inhibitors for Nsp13 [73, 74] 

Nsp5 is also recognized as the 3CLpro and is known for its function to produce mature enzymes upon their automatic cleavage from polyproteins. In addition to their cleavage from the polyproteins, they carry out the cleavage of downstream NSPs at 11 different sites for the Nsp4-Nsp16 release [75] . Asn142 [64] . The structure of the compounds which were reported to target the possible drug target sites present in SARS-CoV-2 is shown in Figure 3. 

The viral RNA synthesis and replication are supported by several other crucial non-structural proteins (NSPs) like Nsp7-Nsp8 complex, Nsp3b, Nsp3e, Nsp9, Nsp10, Nsp14, Nsp15, and 

At present, the host-specific receptor for SARS-CoV-2, i.e., the ACE2 (angiotensinconverting enzyme -2) is contemplated as a potential host-specific target for treating COVID-19 and hindering the entry of SARS-CoV-2 inside the host cells [77] . The sequence of SARS-CoV-2's RBD is somewhat identical to that of the previously encountered SARS-CoV [78] and recent studies including the virtual screenings have made it clear that several drugs like losartan, which is an anti-hypertensive medication, ergotamine (analgesic drug), anti-diabetes drugs like troglitazone, liver-protective druglike silybin and anti-bacterial druglike cefmenoxime, etc., showed low binding energy and high binding affinity with the ACE2 receptors.

Additionally, some studies have demonstrated that targeting and inhibiting the enzyme activity of TMPRSS2, which induces cleavage of S protein and facilitates the CoV Therefore, the rhythmic patterns in the expression and activity profiles of these potential drug targets need to be considered while designing and dosing the candidate drugs.

ORF7a, Nsp1, and Nsp3c are the three main factors that assist the CoVs in evading the immune system and are responsible for meddling with the innate immunity of the host, thus enhancing the virulence of the virus. The release of freshly-assembled CoVs from the host cell is inhibited by bone marrow matrix antigen 2 (BST-2). The activity of BST-2 is inhibited by ORF7a of the CoV as it directly binds to BST-2 and blocks its glycosylation [82] .

Inhibition of the production of type -I interferon and specific degradation of host mRNA is [64] . The possible drugs/therapeutic molecules that may be effective in targeting the essential functional molecules associated with SARS-CoV-2 are enlisted in Table 2 .

The structural elucidation of SARS-CoV-2 reveals several significant functional molecules associated with the virus, essentially required for their survival and pathogenesis in humans. Table 2 : Potential therapeutic molecules/drugs that may be effective in targeting the various essential functional molecules associated with SARS-CoV-2 [64] .

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KR, TR and SR thank IIEST, Shibpur, Howrah, West Bengal, Bihar Agriculture University, Sabour (Bhagalpur) and Mahatma Gandhi Central University Motihari, Bihar, respectively.