Generation of docked poses

The grid for the 6OYH structure was generated using the OPLS3e force field using default parameters. The three prepared ligands, MD1, CPZ-B, and capuramycin, were docked into the grid using default parameters. Although MD1 and CPZ-B docked well into 6OYH with docking scores of −9.88 kcal/mol and −9.49 kcal/mol, respectively, the docking pose of capuramycin was not similar to that of the experimental X-ray crystal structure of MraY from Aquifex aeolicus in complex with capuramycin (PDB ID: 6OYZ). To resolve this issue, we further carried out induced-fit⁴⁴ docking of capuramycin with 6OYH using carbacaprazamycin as the reference ligand. One of the best poses was further refined using the Prime refinement⁴⁵ module (which implements minimization Monte Carlo). The refined docked pose of capuramycin overlaid well with the experimental 6OYH–co-crystallized ligand. These three complexes were used for the generation of e-pharmacophore hypotheses.

Our efforts to dock the three ligands to the MraY_Mtb homology model using SP docking and induced-fit docking protocol proved challenging. Capuramycin could be docked to the receptor using a rigid SP docking protocol. However, for the flexible CPZ-B ligand, we had to devise a different strategy. The carbacaprazamycin ligand was extracted from the experimental X-ray crystal structure of 6OYH and was merged with the aligned MraY_Mtb homology model. The resulting complex was further refined using minimization Monte Carlo to obtain an energy-minimized complex. Subsequently, an induced-fit docking of CPZ-B was carried out with the homology model using the MraY_Mtb–carbacaprazamycin complex as a reference. Gratifyingly, the strategy was fruitful to generate the desired pose of MraY_Mtb–CPZ-B.

Despite our best efforts, MD1 could not be docked into the MraY_Mtb homology model; hence we adopted a different strategy. Since MD1 is an analog of MD2, which is methylated at its C-2” hydroxy of the amino ribosyl moiety (Figure 6), we decided to modify the 2D structure of the MD2 ligand of the MraY_Mtb homology model to form MD1, using the 3D-build suite of Schrödinger. The modified MraY_Mtb–MD1 complex was further refined using the Prime refinement module (minimization Monte Carlo). The complex was then directly used for the e-pharmacophore hypothesis generation.