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Three major outbreaks of acute respiratory syndrome induced by coronaviruses (CoVs) have been witnessed in the last two decades. The first outbreak was Severe Acute Respiratory Syndrome (SARS) in 2003 with Guangdong, China as epicenter (Peiris et al., 2004) followed by Middle East Respiratory Syndrome (MERS) in 2012 in Saudi Arabia (Zaki et al., 2012) and now the novel coronavirus disease (COVID-19), first reported in Wuhan, China in late 2019(Wang et al., 2020). World Health Organization (WHO) declared COVID-19 outbreak as a global pandemic on 11th March 2020 (Cucinotta & Vanelli, 2020). The spread of the disease around the world and the number of infected patients and mortality of COVID-19 are increasing exponentially day by day. As per the WHO report (28th October, 2020), COVID-19 is affecting more than 210 countries and territories with over 43,766,712 confirmed cases and over 1,663,459 total deaths around the world (World Health Organization, 2020). Currently, the global fatality rate is around 3.80% (calculated as deaths per confirmed cases). A large number of people are being identified as COVID positive every day in the USA followed by India, Brazil, Russia, South Africa and other countries.
CoVs are a group of enveloped, positive-sense, single-stranded RNA viruses belonging to the Corona viridae family. They induce respiratory, neurological and gastrointestinal complications of varying severity in human hosts. Novel coronavirus (2019-nCoV) also known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belonging to the category of β-coronavirus is the causative agent of COVID-19 (Chen et al., 2020; Osman et al., 2020).Although SARS-CoV-2 is considered to be introduced from bats, its specific source, animal reservoir and enzootic patterns of transmission remain unresolved.
The SARS-CoV-2 genome, comprising of ~30,000 nucleotides encodes 4 structural proteins, 16 non-structural proteins and 9 accessory proteins (Chen et al., 2020). By translation of the viral genomic RNA (gRNA), CoVs produce two overlapping polyproteins pp1a (450-500 kDa) and pp1ab (750-800 kDa) (Thiel et al., 2003). These polyproteins undergo extensive proteolytic processing and ultimately the functional polypeptides are released which are crucial for the replication and assembly of the virus. This proteolytic processing is mediated predominately by the main protease (Mpro) also referred to as 3-chymotrypsin-like protease (3CLpro) or non-structural protein-5 (NSP-5), and by papain-like protease (PLpro). Mpro is a cysteine protease that digests the polyprotein within the Leu-Gln↓ (Ser, Ala, Gly) sequence (↓indicates the cleavage site), which appears to be a conserved pattern of this protease. The ability of CoVs to hydrolyze the peptide bond specifically after Gln residue is very unique which is unknown for human enzymes (Pillaiyar et al., 2016; Hilgenfeld, 2014). This characteristic feature along with the functional importance of Mpro makes it a potential target for COVID-19 antiviral drug discovery (Zhang, Lin, Sun, Rox, et al., 2020; Jin et al., 2020).
The X-ray crystallographic structure of SARS-CoV-2 Mpro bound to a covalent inhibitor N3 was resolved by Jin et al (2020). Mpro has 306 amino acids long chain with three domains. Domain I contains Phe8 –Tyr101 residues, domain II contains Lys102 –Pro184 residues, and domain III contains Thr201 –Val303 amino acid sequence linked with domain II by a long loop region of Phe185 – Ile200 residues. The substrate-binding site with a Cys145 – His41 catalytic dyad is present in a cleft between domains I and II. The major active subsites where the substrates bind to Mpro are well defined. The S1 subsite is composed of Phe140, Leu141, Asn142, His163, Glu166 and His172 amino acids. A small S1’ subsite involves Thr25, Thr26 and Leu27 residues. Hydrophobic S2 subsite is composed of His41, Met49, Tyr54, Met165 and Asp187 residues. The S4 subsite is made up of Met165, Leu167, Phe185, Gln189 and Gln192 residues (Jin et al., 2020; Zhang, Lin, Sun, Curth, et al., 2020).
The Cys145 amino acid present in catalytic dyad functions as a common nucleophile in the proteolytic cleavage of the natural substrate of Mpro. The proteolytic cleavage is believed to be performed in a multiple-step mechanism (Figure 1). Once the Cys145 side-chain proton is abstracted by the imidazole nitrogen of His41 (Step-I), the resulting thiolate nucleophile attacks the carbonyl amide group of the natural substrate (Step-II). The N-terminal peptide product is released with the abstraction of a proton from His41 (Step-III). Then, the thioester is hydrolyzed (Step-V) and the C-terminal product is released which restores the active catalytic dyad (Step-VI) (Pillaiyar et al., 2016).