# _In Silico_ Development of SARS-CoV-2 Non-Covalent Mpro Inhibitors: A Review
## 2 Mpro Inhibitors for SARS-CoV-2
### Molecular Dynamics (MD) in Drug Development  
Computer-aided drug design (CADD) includes various _in silico_ tools, with molecular dynamics (MD) simulations being one of the most essential methods, particularly in pharmaceutical development and formulation research [86]. Beyond initial hit identification,  
Figure 11: The chemical structures of S5-27 and S5-28. MD simulations enable researchers to assess drug behaviour at the atomic level, providing insights into solubility, conformational flexibility, and complex stability that static docking alone cannot capture. This section explores the significant contributions of MD simulations in developing inhibitors targeting the SARS-CoV-2 Mpro, illuminating how these simulations guide the design and optimisation of promising candidates. MD simulations, by mimicking the dynamic behaviour of protein-inhibitor complexes, allow researchers to probe interactions that are key to inhibitor efficacy. These interactions--hydrogen bonds, hydrophobic contacts, and van der Waals forces--are pivotal in determining how effectively an inhibitor can bind to its target [87]. With this understanding, researchers can predict how modifications to the inhibitor structure may enhance its binding affinity, specificity, and overall therapeutic potential [86]. The true advantage of MD lies in its ability to differentiate between fundamental binding and notable stability under physiological conditions--something often overlooked in research focused solely on docking. Moreover, the progress in machine learning (ML) has increased reliance on MD simulations, streamlining the rapid development of new pharmacological agents. One notable success in the search for effective Mpro inhibitors has been the exploration of compounds derived from withina species. Among them, Withacoagulin H (W30) emerged as a standout candidate [66]. Through 50 ns of MD simulations, it exhibited remarkable stability within the Mpro active site, demonstrating a binding energy calculated by MMGBSA of \(-63.463\) kJ/ mol, significantly outperforming other compounds (Figure 12). This impressive result suggests its potential as a valuable candidate for further in vitro evaluation, bringing it one step closer to becoming a therapeutic option. While MD simulations have shown great promise, many studies have relied on relatively short simulation times (e.g., 20 ns, 50 ns) to predict the stability and binding behaviour of Mpro inhibitors. This is particularly concerning given the inherent flexibility and complexity of the Mpro active site, which may require longer simulation times to capture the dynamic nature of the protein-ligand interactions fully. For instance, the study on W30 reports a 50 ns MD simulation, which, while valuable, might not be sufficient to observe the full range of interactions and potential conformational changes in the complex. The limited duration of these simulations may overlook essential binding events or dynamic behaviours that could influence the inhibitor's long-term stability and efficacy. Moreover, studies combining MD simulations with ensemble docking or simulations extending to microsecond timescales (1 \(\upmu\)s) are rare but crucial for assessing the full extent of binding dynamics. This approach has led to the identification of inhibitors such as Z1244904919 and Z1759961356, whose dynamic binding behaviours were studied in depth with simulations extending to 1 \(\upmu\)s [88]. Binding free energy calculations using the MM/GBSA model showed that electrostatic and hydrophobic interactions are essential for stabilising these complexes, setting the stage for experimental validations. In Table 5, the positive values of \(\Delta\)Gpol indicate unfavourable solvation, which is a standard result in MM/GBSA calculations. These positive \(\Delta\)Gpol values reflect the desolvation penalty for the ligand, meaning that the system resists solvation in the binding process. Thus, polar solvation does not contribute favourably to binding but is instead a hindrance.