Possible mechanism for protein-ligand interaction. Insights to how ligand stabilization occurs within the protein binding site can be discerned from MD simulation. The H-bond formations with Leu224 at 2 and Glu228 at 3 and 4 ''sandwich'' the aminopyridinium and anchors Xylopine_2 ( Figure 6A ). However, the xylopine moiety remained unattached, causing strain to the compound and possibly contributing to large H-bond distance fluctuations ( Figure 7A ). At approximately 15 ns, attraction between Glu228 and 3 causes the terminal amine residue to torque towards Glu228 increasing the distance from Leu224 ( Figure 6A ). As a result, the stable H-bond with Leu224 was lost, and an additional H-bond with Glu228 was formed from 2. At the end of MD simulation, the primary binding force for Xylopine_2 was H-bonds formed with Glu228. In contrast to Xylopine_2, multiple binding sites secured Rosmaricine_14 and 15 within the binding site as reflected by the small H-bond distance fluctuations compared to Xylopine_2 ( Figure 7B , 7C). Rosmaricine_14 bound to Glu119, Glu228, and Arg293 through 6, 7, and 8 respectively ( Figure 6B ). The multiple attachment points anchor both the aminopyridine moiety and the rosmaricine moiety, reducing strain on the molecular structure as reflected by the low total energy and bond distance fluctuations. The increase in bond distance from Arg293 in Figure 7B starting at 3 ns was due to Arg293 intermolecular Hbond formation between the O atom and NH 2 residue. Nonetheless, all H-bonds were maintained throughout the MD simulation, suggesting good stability of Rosmaricine_14. The mechanism for stability of Rosmaricine_15 is similar to that of Rosmaricine_14. In addition to H-bonds at 9 with Glu228 and 10 with Arg293 which are identical to Rosmaricine_14, H-bonds are formed at 11 with Glu228, 12 with Thr226, 13 with Asn262, and 14 with Tyr402 ( Figure 6C ). These additional anchor points stabilized residues that were available for rotation in Rosmaricine_14, further lowering the total energy of the compound ( Figure 5C ). Within the anchored ligand, twisting of 15 contributes to H-bond fluctuations at Glu228 such as Figure 6D ). The stability of Tamiflu as a result of these binding anchors is reflected in the low total energy profile ( Figure 5 ) and small H-bond distance fluctuations ( Figure 7D ). The ability of the de novo derivatives to form stable H1-ligand complexes has also been assessed [92] . All de novo derivatives were capable of forming H-bonds at Glu83 and Asp103, the key binding sites on H1.