Figure . The FES of compounds 1, 4 and 7 binding to plm II obtained using FMetaD simulations and reweighed Poor activity against catD and plm V. The FES of 2-aminoquinazolin-4(3H)-ones binding to plm V (Figure top) and catD (Figure , bottom) are slightly different from those of plm II binding. The majority of the binding modes observed during the inhibitor binding to plm II are present also on the catD and plm V binding pathway, however, there is one noticeable difference -binding under the flap loop is negligible. For both enzymes, the energetically favoured binding mode is similar to that observed in basin B for plm II, where transition state mimetic group is interacting with aspartic dyad while the flap pocket substituent is located between the flap loop and unstructured region right next to it (see compound 7 binding modes B in Figure ). Here hydrophobic flap pocket substituent is interacting with Phe74, Ile76 and/or Val144 of catD, and Tyr286, Tyr288 and/or Leu311 of plm V. Another, similarly favoured binding mode corresponds to the inhibitor bound in a binding groove in the opposite direction, where hydrophobic part of the inhibitor interacts with Phe126 and/or Phe131 of catD, and Leu179 and/or Tyr182 of plm V (see Figure , binding modes A). Despite plm V highly flexible flap loop tip (since Tyr139 is not involved in direct hydrogen bonding with protein core β sheet motif amino acids like plm II and catD, see Figure ) inhibitor binding in a pose where flap pocket substituent is fully buried under the flap loop was not observed. Minor interactions under the plm V flap loop tip (basin C) were similar to those observed in basin C for plm II, where hydrophobic flap pocket substituent was stacked in-between Tyr139 and Phe180 while the transition state mimetic was solvent exposed. The rigidity at the plm V flap loop hinge region does not allow further flap opening, and as such inhibitor remains in non-optimal conformation. Inhibitor interactions under the catD flap loop tip were similar -terminal benzene ring interacted with Tyr78 and Phe126, however it did not lead to inhibitor binding under the flap loop. Similar binding behaviour was observed for all inhibitors. Taking FMetaD simulation data into account, it is safe to assume, that poor plm V and catD inhibition is a result of inhibitor inability to bind under the flap loop, and enzyme inhibition is achieved by inhibitor binding in an alternative pose. These alternative binding modes, however, lack optimal (coplanar) 2-aminoquinazolin-4(3H)-one core placement (due to flap pocket substituent clashing with the plm V and catD flap loops) and, thus, ligand activity is reduced. We believe that structural feature that plays crucial role in inhibitor binding under the flap loop is hydrophobic amino acid region at the flap pocket "entrance" between the flap loop and adjacent α helix. All enzymes have phenylalanine situated at the same place right next to the flap loop tip (Phe111 for plm II, Phe180 for plm V and Phe126 for catD, see Figure ), which in unbound state can interact with flap loop tip Tyr and approaching inhibitor. Such transition states were observed in our simulations, and correspond to previously described states in basin C. Once initial interactions are established, the hydrophobic inhibitor substituent is then guided deeper in the flap pocket to interact with Tyr115 and Trp41 of plm II. In plm II, the Tyr115 sidechain is rigidly locked in the position by hydrogen bonding with Asp121 main chain, whereas Trp41 is known to change the conformation upon non-peptidomimetic inhibitor binding/flap opening. The flap pocket residue rearrangement results in a conformation where hydrophobic flap pocket substituent is locked between Tyr115 and Trp41. The hydrophobic flap pocket remains un-solvated during the flap pocket filling step, and required flap opening is aided by gradual flap pocket filling with hydrophobic inhibitor tail. In plm V and catD, the position of the Tyr115 sidechain is occupied by Glu176 and Gln121, respectively, and, therefore, hydrophobic interactions with the inhibitor are discouraged. This prevents ligand flap pocket substituent guiding deeper under the flap loop and efficient inhibitor binding. The FES of compound 7 binding to plm V (top) and catD (bottom) obtained using FMetaD simulations and reweighed as a function of the ligand core-catalytic dyad distance d1 and a torsion φ. Isosurfaces are shown every 1 kcal/mol. Deepest FES basins for each system are indicated as A and B, and corresponding binding modes are shown on the right. Ligand is shown as green sticks, selected binding pocket residues and catalytic dyad residues as white sticks. Flap loop is in salmon. Hydrogens are omitted for clarity.