Compared to other targets, WaterKit predictions for neuraminidase show a large deviation for E sw values with respect to MD (2.78 kcal/mol, Fig. , and Fig. andS12), but an overall agreement between the WaterKit predictions and the MD results (r 2 0.68 for ∆G, and 0.73 E sw ). This performance is likely a consequence of the instability from the lack of convergence of MD simulations. Nevertheless, the water network predicted by WaterKit shows a good agreement with several key water molecules found in apo structures (Fig. ). Most of the predicted water molecules are found consistently across crystallographic structures and mutants (Fig. ), and their position tend to be occupied by the atoms of the different bound ligands. To analyze the predicted water network, we isolated water molecules identified by WaterKit, as well as crystallographic water molecules from a few representative apo structures (PDB ids: 3nn9, 4nn9, 5nn9, 6nn9, 6crd, 6d3b, 6mcx), both within 1.5 Å from atoms in known binders of neuraminidase. The clusters of conserved crystallographic water molecules trace most of the essential features of sialic acid, the natural enzyme substrate (Fig. ). Wa-terKit results match these features, and extend the network to include water molecules not detected in the crystallographic structures. Two of them, located around the anomeric carbon of the ligand, match the anomeric hydroxyl (W1), and the position of the carbon itself (W2, Fig. ). The position of W2 corresponds marks also the optimal placement for the electrophilic carbon of neuraminidase inhibitors designed to mimic neuraminidase-sialic acid intermediates 152 , and which undergo nucleophilic attack the Tyr406 O η 152,153 . Inhibitors of neuraminidase build upon the sialic acid scaffold by adding chemical moieties aimed at increasing affinity or escape resistance . The analysis of WaterKit results support the hypothesis 156,157 that potent and selective sialic acid analogues, such as drugs zanamivir (Relenza 158,159 ) and oseltamivir (Tamiflu 160 ), as well as the fluorinated derivative FeqGuDFSA 154 , might due their binding affinity by engaging a progressively larger number of water molecules in the predicted water network (Fig. C-E), Similarly, the aromatic inhibitor BANA206 (Fig. ) was designed to establish interactions with conserved water molecules, using a cyclized amide to displace weakly bound water molecules in the vicinity of Ser102 and Glu150. The source of the convergence issues in the MD simulation was not identified. However, it is likelt that given the abundance of charged residues in this pocket, it is not unlikely that by using more accurate electrostatic models in both WaterKit and MD simulations might improve the quality of the results.