Weak forces such as SB431542 H-bonds and hydrophobic interactions play critical roles in the ligand recognition and protein stability, and were analyzed separately. Table 2 summarizes H-bond formation and stability BU 4061T during MD. Aurantiamide formed a single low occupancy H-bond with His280. Cnidiadin formed Hbonds with Tyr131 _and His280. 2-Hexadecenoic acid primarily interacted with Arg128 and Arg273 during MD. Three H-bonds with Arg128 were highly stable with occupancies greater than 91.50%. Orlistat formed stable Hbonds with Gly93, Phe94, Asp96, and Tyr131. Based on the occupancy rate and observation frequency, Phe94 was the key residue for H-bond formation. To account for possible underestimation of H-bond occupancies due to the designated cutoff distance 2.5 A ��, H-bond distance trajectories of each individual H-bond were analyzed. Based on the trajectory shown in Figure 6A, the H-bond distance with His280 generally exceeded the typical H-bond distance of 2.2�C3.2 A ��, indicating that Aurantiamide was probably stabilized within the complex by interactions other than H-bonds. For Cnidiadin, H-bonds at Tyr131 and His280 were within typical H-bond distance ranges, implying that H-bonds formed at these locations were stable and effectual in maintaining stability during MD. Hbond trajectories for 2-hexadecenoic acid at Arg128 show consistent findings to those in Table 2. All six H-bonds detected at Arg128 may contribute to stability albeit some distance being greater than 2.5 A ��. The primary H-bonds formed by Orlistat were with Phe94, Asp96, and Tyr131. Initially, a weak H-bond was formed with Gly93, but was substituted by that with Asp96 at the end of MD. This substitution could be due to conformational changes that increase the distance from Gly93 and decrease the distance from Asp96. Overall, H-bonds were important for the stability of Cnidiadin, 2-hexadecenoicacid, and Orlistat. MD snapshots of the test compounds at 0 ns and 40 ns may help visualize interactions involved in PNLIP-ligand complex stability. As previously mentioned, H-bonds were not a primary stabilizing factor for aurantiamide. The snapshots at 0 ns and 40 ns support this view. At the end of MD, Aurantiamide was anchored within the binding site by pi-interactions with Arg128 and Tyr131 while no H-bonds were observed. Pi-interactions were also involved in stabilizing Cnidiadin during MD. The two piinteractions on opposing sides of Cnidiadin served as invisible chains to anchor Cnidiadin within the PNLIP binding site. These interactions may greatly inhibit ligand movement and contribute to the stable ligand RMSD in Figure 5B. 2-Hexadecenoic acid did not form pi-interactions, and was stabilized through its hydrophilic head region by multiple H-bonds with Arg128 and Arg273. Initially, Orlistat formed only H-bonds, but upon complex stabilization, an additional pi-interaction with Phe232 was observed. Total hydrophobic interactions are also critical for stabilization and the results of Ligplot analysis are shown in Figure 8. The highest number of hydrophobic interactions was observed in Aurantiamide. This was expected as Aurantiamide lacked H-bonds compared to the other test compounds. The hydrophobic contacts primarily interacted with the end cyclohexanes and carbon backbone, providing additional support in addition to the pi-interactions with Arg128 and Tyr131. This balance between interaction forces secures Aurantiamide within the binding site and may be the reason for its low total energy. Six hydrophobic contacts were formed with Cnidiadin and served to stabilize side chains that were not bound by H-bonds and pi-interactions. Hydrophobic contacts were responsible for stabilizing the aliphatic tail of 2- hexadecenoic acid.