**3.2 SARS CoV2 nsp5 Mpro**

The SARS-CoV-2 nsp5 contains 306 amino acids (located in the replicase polyprotein pp1ab, 3264 to 3569 aa), a 33.8-kDa main protease (Mpro), which is also referred to as 3C-like protease (3CLpro) [21, 22]. The active site in SARS-CoV2 Mpro organized in between Domain I (8–99 aa) and Domain II (100–183 aa) was shown to be structurally similar to SARS-CoV Mpro. Both domains contribute one residue to the catalytic dyad (His41 and Cys145), linked to the helical domain IIII (200–306 aa)

#### **Figure 3.**

*Overview of the SARS-CoV2 nsp3 structural and genomic organization along with crystal structure of SARS-CoV-2 papain-like protease (PLpro). (PDB ID: 7D47). Abbreviations: DUF3655, protein of unknown function; macro, macro domain; SUD-N, SARS-unique domain binding G-quadruplexes; SUD-M, singlestranded poly(a) binding domain; PL2pro, coronavirus polyprotein cleavage domain; viral protease, papain like viral protease; NBD, nucleic-acid binding domain; 7tm GPCRs, seven-transmembrane G protein-coupled receptor superfamily; TM helix 5&6, transmembrane helix 5&6.*

*Repurposed Therapeutic Strategies towards COVID-19 Potential Targets Based on Genomics… DOI: http://dx.doi.org/10.5772/intechopen.96728*

by a long loop region (184–199 aa) (**Figure 4**) [6]. Several co-crystalized SARS-Mpro structures bound with inhibitors, 11a and 11b (6LZE), I2 (2D2D), N1 (1WOF), N3 (2AMQ ), and N9 (2AMD), revealed the position of S1, S2, and S4 subsites, especially in the active site close to His41 and Cys145, which is crucial for substrate recognition containing the core sequence [ILMVF]-Q-|-[SGACN] along with Tyr161 and His163 in the substrate-binding pocket [23]. Studies have estimated that the Mpro pp1ab protein has at least 11 conserved restriction sites, beginning with its autolytic cleavage site and even able to bind ADP-ribose-1′ [24, 25]**.**

#### **3.3 SARS CoV2 nsp12 RdRp**

The SARS-CoV-2 nsp12 is a key component of the replication/transcription machinery sharing a strong structural homology with nsp12 of SARS-CoV, indicating that its function and mode of action may be well conserved [26]. SARS-CoV-2 nsp12, is a 106.7 kDa molecular weight protein with a chain length of 932 amino acids (located on the replicase polyprotein 1ab, 4393 to 5324 aa.), also referred to as RNA dependent RNA polymerase (RdRp) [27], has the capacity to synthesize the entire (−) RNA chains as a positive sense RNAs, as templates for additional genomic RNA (gNR) and subgenomic RNAs generation (sgRNAs) of the virus [21]. RNAdependent RNA polymerases (RdRps) are a multi-domain protein that catalyzes polymerization of RNA-template (phosphodiesters formation between ribonucleotides), in the presence of divalent metal ions and thus play a major role in the viral replication and transcription of the SARS-CoV2 [28, 29]. The SARS-CoV-2 nsp12 (S1 to Q932) exists in complex with cofactors nsp7 (S1 to Q83) and nsp8 (A1 to Q198) heteromer with the second nsp8 attached to the distinct binding site. nsp12 contains the right-hand RdRp domain (S367 to F920) and the nidovirus-specific N-terminal β hairpin (D29 to K50) and extension domain (D60 to R249) which adopts the nidovirus RdRp-associated nucleotidyltransferase (NiRAN) architecture (**Figure 5**). The RdRp domain is divided into 3 sub-domains; the finger subdomain (L366–A581 and K621–G679), the palm subdomain (T582–P620 and T680–Q815), and the thumb subdomain (H816–E920) [30]. The nsp7-nsp8 heterodimer binding site is well conserved in the palm domain and overlaps with the functional domains of the preserved polymerase regions (fingers and thumb domains). In addition, seven preserved motifs (A-G) arranged in the polymerase active site domain, involved in template and nucleotide binding and catalysis, were also revealed by SARS-CoV-2 nsp12 structure [31]. Motif A contains the residue of classical divalent ion-binding glutamate (D) in position (618) & (623) of the conserved sequence

#### **Figure 4.**

*Structural representation of SARS-CoV-2 Mpro monomer (PDB ID: 6LZE) (ribbon representation) composed of: N-terminal domain I (cornflower blue), domain II (green), and C-terminal domain III (pink). Substrate recognition site in (green and red) and catalytic dyad residues, His41 and Cys145 are highlighted and labeled.*

#### **Figure 5.**

*Domain organization of COVID-19 virus nsp12 and structural ribbon representation of the SARS-Cov-2 RNA-dependent RNA polymerase in complex with cofactors nsp7-nsp8 (PDB ID: 6M71) displaying domains, conserved motifs (A-G), and protein regions in the structure. A broad N-terminal extension (red) consisting of the NiRAN domain (violet) and the interface domain (cornflower blue) adjacent to the polymerase domain comprises SARS-CoV nsp12-nsp7(orange)-nsp8 (light pink) complex. The interdomain borders are labeled with residue numbers.*

611-TPHLMGW**D**YPKC**D**RAM-626. In motif C, (753- FSMMIL**SDD**AVVCFN-767), the catalytic residues of 759-SDD-761 (amino acids 759,760 and 761 are positioned between two β strands. Many other catalytic residues such as 317-**GDD**-319 and 327-**GDD**-329 are also conserved among other viruses [32].
