# _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
The high concentrations required for darunavir and atazanavir, despite minimal cytotoxicity even at 200 \(\upmu\)m, further underscore the disconnect between protease inhibition and clinical efficacy. These results suggest that the observed antiviral effects might stem from off-target interactions rather than specific Mpro inhibition, emphasising the need for mechanistic clarity and further in vivo validation. Table 2 overviews existing HIV protease inhibitors targeting SARS-CoV-2 Mpro [58]. Natural products have also been extensively explored as Mpro inhibitors. While they offer chemical diversity, many of these studies suffer from an over-reliance on docking scores without a robust experimental follow-up. Table 3 outlines some natural compounds that inhibited Mpro of SARS-CoV-2. For example, shikonin emerged from high-throughput screening (HTS) as a non-covalent Mpro inhibitor with an IC\({}_{50}\) of 0.397 \(\upmu\)m [59]. However, its cytotoxicity limited further development [60]. Similarly, quercetin exhibited modest Mpro affinity, but structural optimisation using selenium-functionalized derivatives enhanced its antiviral activity [61]. Furthermore, selenoquercetin analogues were synthesised and screened as SARS-CoV-2 Mpro inhibitors, demonstrating improved potency over quercetin. The most effective derivative, compound 2d, exhibited an IC\({}_{50}\) of 8 \(\upmu\)m for antiviral activity, representing a 24-fold enhancement compared to quercetin. Consequently, _in silico_ and experimental studies revealed non-covalent inhibition involving a selenium mediated hydrogen bond with Gln189, stabilising the compound within the active site and blocking the His41/Cys145 catalytic dyad [61]. However, although _in silico_ and biochemical data support its binding to the catalytic dyad, its in vivo efficacy and safety remain unproven. Moreover, baicalein, a polyphenolic compound found in _Scutellaria bicalensis_ and other plants, stands out as a more balanced candidate, exhibiting strong docking (\(-9.8\) kcal/mol), low cytotoxicity, and in vitro potency (EC\({}_{50}\) = 1.69 \(\upmu\)m; SI \(>\) 118) [62; 63; 64]. However, detailed pharmacokinetic and metabolic stability studies are needed before clinical translation. Similarly, calycosin-7-O-\(\beta\)-glucopyranoside, derived from natural sources, exhibits strong binding to Mpro with a docking score of \(-8.33\) kcal/mol [65]. This compound, like Baicalein, presents an appealing option for drug development due to its structural characteristics that favour interaction with Mpro's active site, which could lead to effective inhibition of the viral enzyme [65]. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Inhibitor** & **Structure** & **IC\({}_{50}\) (\(\upmu\)m)** \\ \hline Lopinavir+Ritonavir & & 10.9 \\ \hline Ritonavir & & 13.7 \\ \hline Darunavir & & 13.7 \\ \hline Atazanavir & & 13.7 \\ \hline Lopinavir & & 13.7 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Inhibition results in cell culture of HIV protease inhibitors targeting SARS-CoV-2 Mpro [58]. While in vitro potency remains a vital metric, its translation into clinical viability equally relies on pharmacokinetic and toxicity profiles. Shikonin, despite having strong IC\({}_{50}\) values, faced limitations due to cytotoxicity. In contrast, baicalein and calycosin-7-O-\(\beta\)-glucopyranoside displayed promising drug-like behaviour with low toxicity and high selectivity. A broader screening of 73 withanolides using Lipinski's Rule of Five and ADME filters identified several hits with favourable drug likeness [66], but these remain in an early computational stage. While such filters are helpful, they may oversimplify complex pharmacokinetic behaviour, and reliance on them alone can result in false positives or negatives. Rational drug development has contributed to enhanced binding affinity and specificity through methods such as virtual screening of focused libraries (e.g., TuKIC), scaffold hybridization, and the incorporation of favourable pharmacophores (e.g., piperazine rings, selenium atoms).