For the fourth system, BACE1, we chose PDB 6OD6 as input structure. We prepared the protein as described above using OpenEye Spruce. Chain A of PDB 6OD6 was then used for further simulations. The ligand SDFiles from the amide and the biaryl series were selected from previous Janssen reports and had measured bioactivities from the same assay while ligand input files from the spirocyclic series were obtained from the protein-ligand Benchmark set with measured bioactivities from a different assay. The starting binding modes for the different scaffolds were obtained by overlaying the structures onto a crystallographic ligand using the SystemBuilder package as described above. For the amide series, PDB 6OD6 and its crystallographic ligands were used while for the spirocyclic series PDB 4JPC was used as input for the ShapeFit algorithm after align-ing the PDB structure onto PDB 6OD6. PDB 3IN4, aligned onto PDB 6OD6 was used for the biaryl series. Different PDB structures were used in this step in order to perform the structural alignment onto a reference ligand that was most similar to a particular scaffold series. However, all protein-ligand complexes from different series were then prepared for MD simulations using the same protein structure, PDB 6OD6. In the bound state, the BACE1 catalytic aspartates Asp32 and Asp228 were both treated in their ionized forms. For some ligands, multiple potential binding modes had to be considered. For non-symmetric substituted phenyl rings, we performed SepTop calculations between different orientation of the R-groups and the more favorable binding mode was then used for further calculations. The solvated ligand and protein-ligand systems were then created with the Orion floes as described above using the Open Force Field version 2.0.0 52 and AM1-BCC charges for the ligands, Amber ff14SB for the protein, and TIP3P 44 for the water. Sodium and chloride ions were added to reach a salt concentration of 150 mM.