His force analysis and measurements of the minimal distance between ETT and the active site of 09N1, it is clear that the carboxyl group of ETT cannot maintain its interactions with R118, E119 and R371 and 2) the newly derived side chain of ETT cannot be stably accommodated in the 150-cavity. These may induce dissociation of ETT from the active site of 09N1. ETT is a derivative of Neu5Ac2en with difference only on the C-3 position, but cannot stably bind with NA after adding the CX-4945 hydrophobic side group. In the crystal structure, this hydrophobic group points toward the 150-cavity. However, there are no hydrophobic residues inside the 150- cavity in 09N1, so neither hydrophobic contacts nor polar contacts can be formed between ETT and 09N1. In the simulations, ETT was expelled from the binding pocket because of absence of favorable contacts. The above findings ARQ-197 suggested that designing intimate contacts between the derived side group and the residues around the 150-loop is of great importance in making efficient sialic acid derivatives. ZMR was originally designed by replacing the hydroxyl group of Neu5Ac2en with a guanidine group that helped to gain binding affinity through interactions with surrounding acidic residues: the side chains of D151 and E227 and the main chain carbonyls of D151 and W178. In this study, due to its unique chemical properties, ZMR was chosen as the template. Lig 1 was designed by linking ZMR and the fragment that had the best docking score. Our simulations provided evidence that the intimate interaction between the ZMR part of Lig 1 and the active site of NA was well maintained. Moreover, the designed contacts between the derivative part of Lig 1 and the residues around the 150-loop were also maintained very well. Additionally, the role of the flexible linker in between which allows the whole ligand to stretch in a suitable manner is of indispensable importance. All of these approaches guarantee to design a compound with high binding affinity towards group-1 NAs. Although the position of the 150-cavity is just beside the binding pocket in group-1 NAs, the main entrance to the cavity is partially blocked by the side chain of D151. When designing new derivatives based on sialic acid scaffold, angle and length restrictions have to
