To generate the potential binding mode of our hit ligands, we used covalent docking followed by long MD simulations. Briefly, three-dimensional ligand structures (11g and 9b) were generated with LigPrep, using Epik to predict their protonation at pH 7.0 ± 1.0, and diastereoisomeric configurations were derived from the synthesis. The OPLS4 force field was employed for structure generation. Covalent docking was performed with CovDock 53 using Cys25 as an anchor, considering nucleophilic addition to the double bond or triple bond of the aldehyde or nitrile groups as reaction type. For each ligand, up to 10 poses were generated. Poses were selected according to the docking score and relevant interactions.