## 3 Results and Discussion
### Binding Free Energy Calculations from the MD Simulations  
Binding free energy calculations using the MM-GBSA method were performed on frames extracted from each FmlH-ligand complex MD simulation trajectory (Figure 13). The binding free energy of the known most potent co-crystallized ligand was obtained from [PERSON] et al. and plotted for comparison [14]. All the ligands showed average negative binding free energies, indicating favorable binding to FmlH. Hits **2**, **5**, **9**, **24**, and **48** exhibited comparable predicted binding free energies to the most potent ligand (**01**) and hence can be considered as strong FmlH-binding glycomimetics that merit further investigation in the design of FmlH anti-adhesives. The previous study by [PERSON] et al. showed that three molecules with a novel central scaffold (Scaffold 1) exhibited enhanced FmlH binding with improved pharmacokinetic properties [14]. In addition, the study focused on the design and implementation of a novel virtual screening protocol to design FmlH-binding glycomimetics using a hybrid fragment-based e-pharmacophore modeling approach [14]. The hits identified in that study showed thiazole and modified thiazole moieties as Scaffold 1. It was also established that both aromatic and non-aromatic moieties in the central scaffold can be considered as potential approaches to the FmlH-binding glycomimetics design. The hits obtained from Scaffold 1 hopping in our study have shown two distinct kinds of hits: ones with aromatic moieties or with non-aromatic moieties as Scaffold 1 (Figure 4). The non-aromatic Scaffold 1 moieties exhibited hydrogen bonding interactions between the protein residues and the GalNAc-aglycone linker or the peripheral functional groups of the central moiety. The aromatic Scaffold 1 moieties showed a \(\pi\)-\(\pi\) interaction with residue Y46 of FmlH, which was also observed in previous FmlH-binding molecular design studies [14; 18]. This residue is present at the tip of the binding pocket. Additionally, an electrostatic interaction between D45 and the charged center of Scaffold 1 (**16**) was also observed (Figure 4). Residue D45 was found to be important in the glycomimetics binding to FmlH [14; 18]. Several of the hits (**1**-**9** and **39**-**49**) obtained in this study exhibited improved predicted binding compared with the previously reported most potent co-crystallized ligand (**01**), as is evident from their Glide scores. Several of the compounds obtained from Scaffold 1 and Scaffold 2 hopping exhibited a low ADMET risk score of \(\leq\)8, which was much similar to the previously reported compounds **01**, **02**, **03**, and **04**. However, from the set of compounds from Scaffold 2 hopping, five compounds (**40**, **49**, **50**, **55**, and **58**) exhibited a high predicted overall ADMET risk. Our negative control compound (**00**) was a non-galactosidase analogue of the most potent co-crystallized ligand. It has been established that the lectin domain of FmlH (FmlHLD) recognizes the galactose units on the target receptors present on the urinary tract epithelial cells. Without the galactoside moiety, the compound will not bind to FmlH or it will exhibit very little binding. The Glide score of the negative control compound was \(-3.935\) kcal/mol. Figure 13: Average binding free energies of the hit ligands (**2**, **5**, **9**, **24**, **48**) from the MD simulation trajectories, calculated using Prime MM-GBSA. The average binding free energy of the known most potent co-crystallized ligand (**01**), from [PERSON] et al. [14], was included for comparison. Error bars represent the standard error of the mean (SEM). An MD simulation was performed on this negative control-FmIH complex. The calculated binding free energy was \(-18.25\) kcal/mol, a much poorer value than for the hit compounds. Additionally, this compound exhibited several pharmacokinetic risk flags, such as that it is predicted to have a high mutagenicity risk, which was not observed for our other hits.