# _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
Seeking to enhance binding affinity further, researchers introduced structural modifications to Mcule-5948770040 (Figure 7), leading to the development of HL-3-68 and Mcule-CSR-494190-S1, with HL-3-68 showing the strongest binding affinity [49]. However, none of these compounds displayed measurable antiviral activity in cell-based assays. On another front, a piperazine-based inhibitor, GC-14, stood out due to its potent inhibition (IC\({}_{50}\) = 0.40 \(\upmu\)m) and strong antiviral activity (EC\({}_{50}\) = 1.1 \(\upmu\)m), along with low cytotoxicity (CC\({}_{50}\) > 100 \(\upmu\)m) and high selectivity for SARS-CoV-2 Mpro [50]. These findings highlight the ongoing challenges in balancing potency, cytotoxicity, and antiviral efficacy in the search for effective Mpro inhibitors. Compound 26 and compound 18 demonstrate that high enzymatic potency does not guarantee antiviral efficacy, particularly when cytotoxicity (as seen in compound 26) or poor cellular uptake (as likely in compound 18) limit their utility. This pattern recurs with HL-3-68, which fails in cellular assays despite its strong binding affinity--emphasising the well-known disconnect between in vitro target engagement and whole-cell antiviral efficacy. Screening an ultra-large set of 235 million molecules led to the selection of imidazolidine-2,4-dione-based inhibitors, which were further optimised to develop compound 19 with sub-micromolar inhibitory activity and broad-spectrum anti-coronavirus properties [51]. Similarly, high-throughput screening (HTS) using FRET assays uncovered non-covalent inhibitors such as walrycin B [52] and quinazoline derivative QZA [53]; however, their therapeutic potential was limited due to toxicity concerns. In contrast, masitimib, identified \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Inhibitor** & **Structure** & **Strategies for the Discovery of Non-Covalent** & **Mpro IC\({}_{50}\) (\(\upmu\)m)** & **Ref.** \\  & & **Mpro Inhibitors** & & \\ \hline Z-DQMD-FMK & & Machine learning-driven virtual screening. & 0.92 & [55] \\ Z4927220858 & & Deep docking methods. & \(\sim\)10 & [56] \\ \hline \hline \end{tabular}
\end{table} 
Table 1: _Cont._  
Figure 7: Structural modifications of the hit compound (Mcule-5948770040) [41]. through cell-based screening, exhibited vigorous anti-SARS-CoV-2 activity (EC\({}_{50}\) = 3.2 \(\upmu\)m) and was validated structurally as a Mpro inhibitor [54]. Machine learning-driven virtual screening enhanced efficiency, ultimately leading to the discovery of Z-DQMD-FMK, which exhibited an IC\({}_{50}\) of 0.92 \(\upmu\)m for Mpro inhibition [55]. Likewise, deep docking methods identified Z4927220858 (IC\({}_{50}\) -10 \(\upmu\)m) spanning multiple Mpro subsites, further emphasising AI's role in improving hit rates [56]. Compounds such as GC-14 and Masitinib exhibit sub-micromolar inhibitory activity and present favourable selectivity and cytotoxicity profiles, critical parameters for clinical development. To discover new inhibitors, a set of thioester-based inhibitors was designed via virtual screening from the Tubingen Kinase Inhibitor Collection (TuKIC) and exhibited notable inhibitory activity against Mpro, supported by co-crystal structures that revealed key interactions [57]. While this study is commendable for experimentally validating hits through crystallography, it stops short of evaluating these compounds in relevant cellular or in vivo models, leaving questions about pharmacokinetics, bioavailability, and toxicity unanswered. Figure 8 summarises the synthesis and activity of these thioesters, such as compounds 3w and 3x, which showed potent inhibition in vitro. In contrast, repurposing efforts using HIV protease inhibitors like lopinavir and ritonavir initially showed promise in vitro, with lopinavir/ritonavir yielding the lowest IC\({}_{50}\) value. However, these compounds also significantly impaired cell viability [58], suggesting a narrow therapeutic index and raising doubts about their suitability for treating COVID-19.