In case A, the poses obtained had carbamate alkyls oriented towards the acyl-binding pocket, while in case B, the poses obtained had carbamate alkyls oriented towards the choline-binding pocket (Figure ). To decide which solution was more probable, binding pose metadynamics (Figure ) and 1 µs all-atom molecular dynamics (MD) simulations were performed for both cases. The metadynamics suggested that case A pose is more stable; however, extended MD simulations revealed that both poses were unstable, as evident from the observed RMSD increase, broken hydrogen bonds with oxyanion hole residues, and the disruption of the catalytic triad (Figure ). Namely, visual inspection revealed that in both cases, the carbonyl of the carbamate moiety that had resided in the oxyanion hole underwent a 180 ° rotation (after 50 and 250 ns, respectively), and that the molecule distanced itself from the catalytic Ser198, thus preventing a covalent reaction. This instability of the prereaction poses suggests that this ligand does not favour the short-lived non-covalent position, consistent with the experimentally observed non-covalent inhibition for this compound. It should be however noted that other factors, such as small changes in the protonation pattern of the charged binding site residues, the force field parameters used and the use of the docking approach (which was optimised for non-covalent binding avoiding steric conflicts) might also be important for the observed outcomes. The results of the in silico covalent and non-covalent binding experiments indicated that the bound orientation described by case A (where the carbamate alkyl is oriented towards the acylbinding pocket) is more probable for this series of compounds. Consequently, all of the remaining 7-substituted indolyl carbamates were modelled in this way. From the pool of non-covalently docked molecules of this series, only 9, 12, 18 and 20 with bulkier N-dialkyl substituents on the carbamate warhead produced no successful prereaction docking poses. Apart from 18, these compounds showed time-independent profiles of reactivity in the in vitro assay, suggesting reversible binding only, which is in agreement with the docking results. Furthermore, the covalent docking protocol successfully produced postreaction poses for all compounds except 12 and 20, both with bulky carbamate warheads and exhibiting timeindependent behaviour in the in vitro assay. The analysis of MD simulations of postreaction poses for time-independent, reversible 9 (Figure ) showed that both poses were comparably stable within the given timeframe and exhibited a similar interaction profile.