Probing into the Flap-dimer Dynamics of the Kasa Enzyme Binding Landscape Provides the Underlying Inhibitory Mechanisms of JSF-3285 and 5G.

Background: β-ketoacyl-ACP synthase I (KasA I) enzyme is crucial in mycolic acid synthesis catalytic condensation reactions, hence implicated in 's virulence and drug resistance. Presently, there is no known potent KasA inhibitor; thiolactomycin lacks potency. Recently reported indazole compounds JSF-3285/DG167 and 5G/DG167 inhibit the KasA through binding to the substrate cavity. However, the molecular mechanism is still unclear, and the unknown resistance mechanisms raise concerns about JSF-3285's novelty.

Methods: This study is the first to report the flap dimer opening and closing of the KasA pocket using combined metrics to define the symmetry impact of the flap-dimer motions and investigate the underlying inhibitory mechanism of DG167 andDG167 using all-atom MD simulation.

Results: The distance/ between the flap (PRO147) and dimer (LEU205) residues; TriC-α angle (: PRO147-VAL83-LEU205 & : PRO147-GLU199-LEU205); and the dihedral angle () were applied to investigate the flap "twisting" and dimer shift closing due to concerted motion by adjacent glycine-rich and glutamic acid-rich loops around the active site during the binding pocket's opening. The full flap-dimer of the unbound opens at 230 ns (d1 = 21.51 Å), corresponding to the largest TriC-α angle 44.5° as is unreliable to describe the flap-dimer motion. The overall averages and for the bounds were ~23.13° and ~23.31°, respectively. Thus, the degree of KasA flap dimer opening is best investigated by distance and . BFE (Kcal/mol) of -44.05 (DG167) showed a higher affinity for the pocket than tr2DG167-KasA (-32.16). Both DG167 and DG167 formed hydrophobic interactions with LEU116, GLY117, ALA119, and tr1DG167 formed strong H-bonds with GLU199. The average RMSD of 2.80 Å (Apo) and RoG of 20.97 Å showed that KasA is less stable and less tightly packed without the inhibitors.

Conclusion: These findings provide a background for a new structure-based design of novel KasA inhibitors.