# _In Silico_ Development of SARS-CoV-2 Non-Covalent Mpro Inhibitors: A Review
## 1 Introduction  
### The Main Protease (Mpro) Enzyme of SARS-CoV-2  
The World Health Organisation (WHO) defines the coronavirus that causes severe acute respiratory syndrome (SARS-CoV-2) as COVID-19 [1]. Recently, the virus has become a global threat [2]. With a genome size of about 30 kb, it is a highly contagious positive-strand RNA virus that codes for 29 distinct proteins essential to the virus's survival and life cycle [3]. The beta coronavirus, or SARS-CoV-2, is closely linked to the SARS-CoV virus that caused the outbreak of the disease in 2002 and 2004 [4]. The coronavirus SARS-CoV-2 is a large, enveloped virus with a single-stranded, non-segmented, positive-sense RNA genome, belonging to the _Sarbecovirus_ subgenus of the _Betacoronavirus_ genus, within the _Coronaviridae_ family. It primarily infects humans but is also known to infect various mammalian species [5]. As shown in the genomic organisation of the SARS-CoV-2 virus in Figure 1, the virion contains both non-structural proteins (nsps) and structural proteins (S, E, M, N) along withseveral accessory proteins that are essential for its replication, assembly, and infection processes [6]. SARS-CoV-2 has four primary structural proteins: spike (S) glycoprotein, small envelope (E) glycoprotein, membrane (M) glycoprotein, and nucleocapsid (N) protein [7]. These structural proteins support the viral entry and assembly processes [3]. The spike (S) protein facilitates viral entry into host cells by binding to ACE2 receptors on the host's cells [8]. The nucleocapsid (N) protein is involved in packaging the viral RNA genome and plays an essential role in viral replication [9]. Additionally, the membrane (M) protein interacts with other structural proteins to determine the shape of the viral envelope, while the envelope (E) protein contributes to viral production and maturation [10]. However, beyond these structural components, SARS-CoV-2 also expresses several non-structural proteins (nsps) that are critical to the virus's ability to replicate and survive within host cells [10]. Unlike structural proteins, which build the virus's physical structure, nsps are responsible for the enzymatic and regulatory functions of the viral life cycle [12]. These proteins are initially translated as part of the large ORF1 ab polyprotein, which is cleaved by two viral proteases--the main protease (Mpro or 3 CLpro, 3-Chymotrypsin-like Protease or nsp5) and the papain-like protease (PLpro)--to release 16 individual nsps [13]. Each of these proteins performs specialised functions that collectively form the virus's replication and transcription complex (RTC), essential for SARS-CoV-2's pathogenicity [14]. Key nsps include nsp12, the RNA-dependent RNA polymerase (RdRp), which synthesises the viral RNA, and nsp13, a helicase that unwinds RNA, thus supporting efficient replication [15]. Nsp3, containing PLpro, plays a dual role in both cleaving viral polyproteins and modulating host immune responses [16]. The main protease, nsp5 (Mpro), is particularly significant, as it processes the majority of viral polyprotein cleavage sites, making it a prime target for antiviral drugs [17]. Like other coronaviruses, these proteins enclose the RNA genome [5]. The main protease (Mpro), the papain-like protease (Plpro), and the RNA-dependent RNA polymerase (RdRp) are the only three major targets for drug development against the SARS-CoV-2 virus [18]. Mpro is a cysteine protease that plays a critical role in the replication of SARS-CoV-2. Notably, it shares approximately 96% sequence and structural similarity with the Mpro of SARS-CoV, as illustrated in Figure 2A. Despite differences among coronaviruses, Mpro demonstrates intense sequence and structural conservation, distinguishing it from human proteases and making it an attractive target for drug discovery to inhibit viral replication and proliferation [19]. Functionally, Mpro operates as a homodimer composed of two protomers [9]. Structurally, Mpro consists of three domains.