# _In Silico_ Development of SARS-CoV-2 Non-Covalent Mpro Inhibitors: A Review
## 2 Mpro Inhibitors for SARS-CoV-2
### Discovery Strategies for Non-Covalent Inhibitors of Mpro  
Unlike covalent inhibitors, which form irreversible bonds with the catalytic dyad and which may raise concerns about off-target toxicity and resistance, non-covalent inhibitors engage the active site reversibly through hydrogen bonding, hydrophobic contacts, and van der Waals interactions. These interactions often lead to improved selectivity, reduced toxicity, better pharmacokinetic profiles, and a lower likelihood of resistance development [16]. Based on recent findings, four key guidelines have been established to design highly effective inhibitors targeting the SARS-CoV-2 Mpro enzyme. First, Rule 1 highlights that the S1 sub-pocket requires a ring of five or six members with a proton donor or acceptor (-NH or =O group) to form crucial hydrogen bonds with a particular residue. Moving to Rule 2, in the S1\({}^{\prime}\) sub-pocket, which is known for hydration and design issues, it is critical to establish a water bridge between the scaffold and Thr26, as well as hydrogen/covalent bonding with the Asn142-Gly143-Ser144-Cys145 (NGSC) motif. Furthermore, the S2 sub-pocket favours cyclic moiety interactions with His41 or hydrogen bonding with His41 and Gln189, which increases efficacy. Finally, the S3 binding site requires a significant aromatic or aliphatic component. It has been observed that while the S1 and S2 sub-pockets are significant for noncovalently bound inhibitors, the S1\({}^{\prime}\) and S3 sub-pockets may influence inhibitor efficacy [42]. Considering these structural insights, ensitrelvir (S-217622) was approved in Japan in November 2022 under the brand name Xocova(r). Its discovery involved a combination of virtual screening, biological assays, and structure-based drug design [17]. As shown in Figure 5, optimising the P1\({}^{\prime}\) ligand and introducing a 6-chloro-2-methyl-2H-indazole scaffold enhanced its inhibitory activity by 90-fold, achieving an IC\({}_{50}\) of 0.013 \(\upmu\)m and an  
Figure 4: Multiple Mpro covalent inhibitors targeting SARS-CoV-2 [37]. EC\({}_{50}\) of 0.37 \(\upmu\)m. The compound demonstrated broad-spectrum antiviral activity against SARS-CoV-2 variants and favourable pharmacokinetics for oral dosing, including high metabolic stability (96% in human liver microsomes) and 97% oral absorption. Clinical trials confirmed its safety and tolerability, with no significant safety concerns identified, leading to both emergency and full approval in Japan [43]. Meanwhile, mprosevir (WU-04), discovered through DNA-encoded library screening, utilises an isoquinoline-based core to inhibit SARS-CoV-2 Mpro non-covalently with an IC\({}_{50}\) of 0.072 \(\upmu\)m [44]. Preclinical studies showed antiviral efficacy comparable to nitrna-trelvir in animal models [45]. Phase 1 clinical trials evaluated its safety, tolerability, and pharmacokinetics, whereas Phase 2 and ongoing Phase 3 trials aim to establish its efficacy in mild-to-moderate COVID-19 cases [34]. The lack of published Phase 2/3 data limits direct comparison with ensitrelvir. Another noteworthy Mpro non-covalent inhibitor is ML188, identified through structure-based drug design, binds the active site with an IC\({}_{50}\) of 1.5 \(\upmu\)m, interacts with the catalytic dyad (His41 and Cys145) via hydrogen bonds, and stabilises the S1\({}^{\prime}\) subsite through hydrophobic interactions [32]. Given its promising binding characteristics, ML188 is an essential scaffold for further optimisation. Figure 6 represents the chemical optimizations of ML188, which yielded compound 23R with an IC\({}_{50}\) of 0.20 \(\upmu\)m, demonstrating significantly improved potency. Moreover, it exhibited antiviral activity in both Vero E6 (EC\({}_{50}\) = 1.27 \(\upmu\)m) and Calu-3 cells (EC\({}_{50}\) = 3.03 \(\upmu\)m). Structural analysis using X-ray crystallography revealed that 23R induced a novel binding pocket between the S2 and S4 subsites, contributing to its strong interaction with the enzyme [46].