## 3 Results
### Docking-Based Screening  
To discover novel Mtb FAS-I inhibitors, the cryo-EM structure of FAS-I in complex with FMN (PDB ID: 6 GJC) was used for molecular docking-based virtual screening utilizing the Surflex-dock module of the Sybyl 2.1 software. The NADP binding pocket was used for performing docking-based screening, comprised of residues Met435, Thr436, Pro437, ValL440, Ala458, Gly460, GLY583, His584, His585, Ala693, Asp694, Ile695, Pro852, Arg868, Ser869, Asp870, Ser871, Leu872, Trp873, Gln874, and the FMN molecule. To address accuracy and efficiency, we carried out the screening protocol in a hierarchical strategy summarized in a work-flow diagram (Figure 2).  
\begin{table}
\begin{tabular}{p{113.8 pt} p{113.8 pt}} \hline \hline
**NADPH Binding Site Residues from Thermomyces Lanuginosus (PDB Code: 4V59)** & **NADPH Binding Site Residues from Mtb (PDB Code: 6 GJC) Obtained by Docking** \\ \hline \hline Tyr636 & Tyr495 \\ \hline Asp668 & - \\ \hline Asp952 & - \\ \hline Lys1026 & Lys851 \\ \hline Pro1027 & Pro852 \\ \hline Asp1045 & Asp870 \\ \hline Ser1046 & Ser871 \\ \hline Lys1044 & Ser869 \\ \hline Leu1047 & Leu872 \\ \hline Thr609 & Thr436 \\ \hline Ile663 & Leu490 \\ \hline Gly749 & Gly582 \\ \hline Gly750 & Gly583 \\ \hline His751 & His584 \\ \hline Glu863 & - \\ \hline \hline \end{tabular}
\end{table}
Table 1: The residue comparison for the NADPH binding site of enoyl reductase between fungal and Mtb FAS-I.  
Figure 1: (**a**) Mtb FAS-I shown in cyan color with bound FMN and docked NADP (**b**) The enoyl reductase domain of Mtb FAS-I (cyan color, PDB:6 GJC) superimposed onto Thermomyces lanuginosus FAS-I (pink color, PDB:4V59).  
Primary screening: The Maybridge screening library consisting of 54,646 molecules was docked using Surflex-dock into the active site of Mtb FAS-I. The top scoring molecules with a score equal to or higher than the score (8.0) for the binding site were considered for the next step. The choice of cut-off value in this study was guided by reproduction of docking pose of phenylimidazole derivative inhibitor enoyl-ACP reductase (FabK) from Streptococcus pneumoniae, which is competitive inhibitor of NADPH (PDB:2Z6). This cut-off resulted in 528 molecules, which were docked again using Surflex-GeomX mode for re-ranking and to improve pose accuracy. Subsequently, the top 150 molecules were inspected visually for favorable interactions.  
Visual inspection: The hits from GeomX were inspected visually for their binding mode(s) for further selection. The following criteria were considered: (1) \(\pi\)-\(\pi\) stacking interaction between the ligand and the FMN molecule; (2) the interaction with residues His584 and Thr436; (3) the formation of hydrogen bonds and other hydrophobic interactions; and (4) the stability in docked pose and fitness of molecule in the binding site. This step was primarily used to enhance specificity and eliminate the compounds having higher score due to interactions with other residues. Based on these criteria, a total of nine molecules were selected for subsequent molecular dynamics simulations. The structures of these compounds are shown in Figure 3.  
Figure 2: The work plan for molecular docking-based virtual screening.  
Figure 3: The structures of nine selected compounds after docking-based screening are shown along with the inhibitor of \(S\). _pneumoniae_ Enoyl-Acyl Carrier Protein Reductase (FabK). The labels of four putative inhibitors are highlighted in boxes.