# Lessons Learnt from COVID-19: Computational Strategies for Facing Present and Future Pandemics
## 1 The COVID-19 Pandemic
### Main Protease (3 CL\({}^{\prime\prime\prime}\))  
Considering the uncertainty about the efficacy of existing treatments [133] and booster vaccinations [134] against present and future Omicron subvariants, the need to find more reliable and variant-agnostic therapeutic tools against COVID-19 is emerging. The previously mentioned issues with the continuously mutating spike protein, which affects most present gold-standard COVID-19 treatments, indicate that different viral targets should be explored for developing novel antiviral drugs [135]. Generally speaking, an ideal target would have to play a pivotal role in the virus replication cycle and be highly conserved across different viral strains [136]. Within SARS-CoV-2, this role is portrayed by its main protease [137] (M\({}^{\text{pro}}\), or 3C-like protease / 3 CL\({}^{\text{pro}}\) due to similarities with the picomavirus 3C protease [138]), thanks to its conserved fold across different coronaviruses [138; 139; 140; 141] (including SARS-CoV [142]) and essentiality for the replication of this virus's subfamily [143]. SARS-CoV-2 MP\({}^{\text{pro}}\), also called nsp5, is a cysteine protease composed of 306 residues [144] that steers the maturation of two partially overlapping polyproteins (pp1a and pp1 ab) into individual mature nonstructural proteins (including M\({}^{\text{pro}}\) itself) through their proteolytic cleavage [145]. Functionally speaking, M\({}^{\text{pro}}\) exists in equilibrium between a monomeric and a homodimer form [146; 147; 148]. This dimerization directly influences the shape of the catalytic site [147], thus altering the enzymatic activity [138] and playing an indirect regulatory role during the virus replication cycle [149; 150]. Within the M\({}^{\text{pro}}\) functional dimer, each protomer is composed of three structural domains. The chymotrypsin-like fold, including \(\beta\)-barrel domain I (residues 1-99) and II (residues 100-182), hosts the active site and thus has direct control over the catalytic event [138; 147], while the \(\alpha\)-helical domain III (residues 198-306) is mainly involved in the direct regulation of dimerization, exerting only a secondary and indirect role on regulating M\({}^{\text{pro}}\)'s enzymatic activity [151]. Between the second and third domains lies a flexible 16-residue loop (residues 183-197) [152]. As anticipated, the catalytic site is located between domains I and II, bordered by the N-terminal domain I of the second protomer in the dimer (Figure 2). Notably, the N-finger (residues 1-7) interacts with the binding site through a salt bridge between the positively charged end of Ser1 and the negatively charged end of Glu166 [153]. The latter is also involved in forming a hydrogen bond with His172, an essential interaction for the enzyme's proteolytic activity [154]. These interactions are so crucial in stabilizing the catalytic site [155] that N-finger deletion impairs dimerization and abolishes the protease's enzymatic activity [156]. Figure 2: **(A)** Crystal structure of SARS-CoV-2 MP\({}^{\text{pro}}\) (PDB ID: 6Y2E): the first protomer is colored in salmon, while the second protomer is colored in pink, and the active site position is highlighted with a blue circle. **(B)** Close-up view of the catalytic site: the H41-C145 dyad is highlighted, alongside the conserved water molecule that substitutes the third member of the canonical catalytic triad diffused in several cysteine proteases. M\({}^{\rm pro}\)'s shallow, plastic, and solvent-exposed active site [152; 157] comprises several subpockets (ranging from S6 to S3\({}^{\prime}\)), hosting the corresponding substrate residues (which vary from P6 to P3\({}^{\prime}\)) [139]. Speaking of substrates, the SARS-CoV-2 M\({}^{\rm pro}\) cleaves peptide bonds at the C-terminus end of a glutamine residue (P1) [137], which is conserved across different SARS-CoV-2, SARS-CoV, and even MERS-CoV substrate sequences [152].