Schematic representation of the funnel shaped restraint potential used in our FMetaD simulations is given in Figure .A. The restraint potential was placed in a way that does not interfere with ligand binding and unbinding events, and its dimensions were chosen as small as possible to accelerate convergence, but large enough to avoid artefacts in the sampling. The funnel defining parameters are summarised in Figure .A. Here, the CV controlling the ligand binding/unbinding was the distance (d1) between the centre of mass of ligand transition state mimetic atoms and the centre of mass of the aspartic dyad Asp34 and Asp214 Cγ atoms (see Figure ). Distance CV easily discriminates between bound and unbound states, however, trajectory obtained can be reweighed to remap the free energy as a function of alternative CVs and gain more detailed insights, revealing bound minima, which are overlying in the maps corresponding to the initial CV choice . Several unbiased and exploratory MetaD simulations were first performed to get an idea of the preferred path of unbinding with statistical significance. During our preliminary simulations we observed that flap loop does not close completely upon ligand unbinding, and rotation of the Trp41, which is known to change conformation when bound to open-flap inhibitors (see Figure .A), was not observed. To overcome this obstacle, we introduced PathCV that allows to study transitions between two or more states. Here we constructed the flap loop opening/closing pathway (including Trp41 rotation) by running trial Path MetaD simulations using X-ray crystal structures of closed and open flap conformations as end states (PDB IDs 1LF4 and 4Z22, respectively; see Figure ). Equally spaced loop opening/closing pathway was constructed from the preliminary simulations and was further used in ligand binding/unbinding studies. Additionally, soft harmonic restraining walls were applied at both ends of the funnel axis to limit the exploration of the ligand inside the region enclosed by the funnel potential. The overall structure of the protein was preserved throughout simulations, during which the loops and the binding site residues showed the necessary flexibility to allow binding and unbinding of the open-flap inhibitors. Binding to plm II. The performed metadynamics simulations directly provide an estimate of where the system spends most of its time . The FES computed as a function of distance d1 and torsion φ (ligand alignment with respect to flap pocket axis, see Figure ) indicate that there are several stabilised transition states along the ligand binding pathway (see Figure ). Most of these states are observed for all of the plm inhibitors synthesized and tested. The deepest basin A in all simulations corresponds to the state where inhibitor is bound in a pose analogue to that found in crystal structure (PDB ID: 4Z22). The 2-aminoquinazolin-4(3H)one core that mimics the transition state is involved in hydrogen bonding with Asp34 and Asp214, adjacent ligand core aromatic ring is interacting with the flap loop Tyr77 sidechain, whereas flap pocket substituent is buried under the flap loop. The dominant interactions under the flap loop depend on the flap pocket substituent length, with longest substituents interacting with Val105, Phe111 and/or Tyr115 sidechains deeper in the flap pocket, while shorter inhibitor substituents are interacting with Trp41, Phe111 and/or Phe120 sidechains. In all cases, ligand binding have disrupted the hydrogen bonding between Trp41 and Tyr77, which is common for aspartic proteases in closed flap conformation . Once this interaction is not present, enzyme flap loop is able to open-up and Trp41 is able to rotate to expose the hydrophobic depth of the hydrophobic pocket. This behaviour is observed upon binding of all inhibitors, even those with short flap pocket substituents. It is worth noting that Tyr77 stacking interactions with inhibitor core in basin A were observed on both sides of the inhibitor (e.g. inhibitor locked in-between Tyr77 and Phe111, or inhibitor interacting with Tyr77 on one side and solvent exposed on the other). Basin B corresponds to the binding mode where ligand core (2-aminoquinazolin-4(3H)-one group) is occupying pose similar to that of crystal structure, while the flap pocket substituent is not buried under the flap loop. Flap pocket substituent here is interacting with Tyr77 and Trp41, however, it is situated on the other side of these residues and is partly solvent exposed. Since this binding mode is observed when the flap loop is in semi-open conformation, we believe that this might be one of the transition states in the ligand binding and flap loop opening pathway. More specifically, the flap loop opening is initiated by the inhibitor binding in the substrate binding groove, and is followed by the inhibitor rearrangement were hydrophobic flap pocket substituent is guided underneath the flap loop while maintaining interactions with aspartic dyad. During this flap opening motion the common aspartic proteases hydrogen bonding network is disrupted and Trp41 rotates to expose the terminal end of the hydrophobic flap pocket and engage in aromatic stacking interactions with flap pocket substituent. Besides previously described poses, where ligand core is interacting with catalytic dyad, two additional binding modes were identified for majority of the inhibitors. One, where aromatic group of the hydrophobic flap pocket substituent is wedged in-between Tyr77 and Phe111/Tyr115 while ligand core is solvent exposed (basin C in Figure ), and another one, where the inhibitor is interacting with aromatic residues Phe11, Tyr17 in the binding grove (basin D in Figure ). Ligand-protein interactions observed in basin D take place ~2 nm away from the aspartic dyad, therefore, it is unlikely that they play crucial role in inhibitor recognition and inhibitor binding, however, these intermediate states might play role in ligand guiding to the binding site. Basin C corresponds to the state where inhibitor is situated closer to the binding site, however, does not interact directly with the aspartic dyad. Interactions here are dominated by the hydrophobic stacking between the inhibitor terminal benzene ring and Tyr77, Phe111 and/or Tyr115 sidechains in flap loop and α helix right next to the loop. These interactions are mostly observed when the flap loop is in closed conformation, and just like complex in basin B, could be a transition state on the flap loop opening pathway. Here flap loop opening is initiated by the hydrophobic interactions that direct the flap pocket substituent under the flap loop by successively interacting with aromatic residues Phe120, Tyr115, Phe111, Tyr77 and finally Trp41, while the transition state mimetic remains solvent exposed. The presence of alternative binding modes observed in FMetaD simulations is supported by the X-ray diffraction data. Attempts were made to co-crystallise plm II and compounds synthesized (see SI for details), however, resolution for the majority of the crystals obtained were suboptimal. The only crystal that was solved was with compound 7 (PDB ID: 7QYH, to be released upon publication). Due to poorly defined electron density in the plm II binding site, it was not possible to solve the position of compound 7, however, unmodelled electron density in the binding site indicates ambiguous flap pocket substituent binding (see Figure ). Electron density map suggest that most probable phenlypentyl substituent placement is under the flap loop on either side of the Trp41, which matches FES basins A and B observed in simulations. The states described above are observed for all inhibitors, however, there are some notable differences between inhibitors. The FES of the 2-aminoquinazolin-4(3H)-one without flap pocket substituent ( ) is the smoothest one, indicating that the inhibitor binding energy is low and energetic penalty associated with the transition from one binding mode to another one is also low (~2 kcal/mol). Several energetically similar binding modes with negligible transition energies mean that the inhibitor is highly mobile and fluctuates between multiple competing binding modes, and, as a result, its inhibition potency is low. Introduction of more extensive flap pocket substituent reduces the accessible binding space and shifts the binding preference to the binding mode observed in the crystal structure. This results in improved inhibitor potency, since role of the alternative binding modes is diminished. This is in line with experimentally determined activity data, where inhibition potency increases as flap pocket substituent length increases, however, the impact of the flap pocket length increase is not linear. We believe that lower activity for compounds bearing phenylmethyl-and phenylethylsubstituents (2, 3) is a result of sub-optimal terminal benzene ring placement in the flap pocket due to competitive stacking interactions. More specifically, shorter linker length prevents simultaneous ligand core stacking interactions with Tyr77 and/or Phe111, and terminal benzene ring interactions with Trp41 and/or Tyr115. Thus, compounds bearing methylene and ethylene linkers are able to form optimal interactions either with catalytic dyad and Tyr77/Phe111, or with Tpr41/Tyr115, but not both. In addition, inhibitors with shorter flap pocket substituents bind to the open flap conformation, however, they are not able to fill the pocket completely, thus, leading to partly solvated hydrophobic pocket and/or introducing thermodynamic penalty due to partly filled cavity. The increase of the linker length enables simultaneous interactions for both groups, reaching optimal positioning with pentylene linker (comp. 7). Moreover, the phenylpentyl substituent size is optimal to fill the flap pocket completely. Flap pocket substituents bearing longer alkyl chain linker can fit in the cavity (8, 9), however, substituents that are too long in their fully extended conformation are expected to adopt energetically unfavourable conformations with internal gauche strains, leading to reduced potency . The potency of the inhibitors with fixed flap pocket length and various benzene ring position (11-15, 7) indicate that the flap pocket depth filling improves the inhibitor activity to sub-micromolar level. The fact that SAR with respect to benzene ring position for this inhibitor subset was identical to initial inhibitor subset indicate that aromatic stacking and/or steric effects provided by the benzene ring play the same, decisive role in inhibitor activity. The additional terminal n-alkyl substituent for this inhibitor series improves the overall activity with respect to corresponding shorter substituents by filling the depth of the flap pocket, thus, reducing the thermodynamic penalty due to unfilled and/or partly solvated hydrophobic pocket .