Metadynamics simulations (MTD) were performed and the free energy landscapes of LysoPS(18:1) and its derivatives (trans/cis-1a-2p and trans/cis-1p-2a) were constructed with respect to the dihedral angles CV1 and CV2 (CV1: green, corresponding to the dihedral angle 1 (D1) for REMD; CV2: orange, corresponding to the dihedral angle 2 (D2) for REMD) (Figure ). Energy landscapes were obtained at 300K (Figure ), and red to blue colors represent low to high free energy states. For LysoPS(18:1), the minima MTD was also carried out for the four cyclic LysoPS derivatives and the corresponding landscapes were obtained (Figure ). The minima of trans-1a-2p are located at around 60° and 180° along the CV1 axis (lower than 5.0 kcal/mol, red region), while the free energy of the regions along CV1 from -180° to 0° is extremely high (20.0 kcal/mol) because cyclized glycerol blocks single bond rotation (white region, Figure ). Similarly, the minima (lower than 5.0 kcal/mol) for cis-1a-2p (red regions) along CV1 were located around ±60°, and very high free energy states (white color region) were located around ±180° to ±120° (20.0 kcal/mol) along CV1 (Figure ). Both trans-1a-2p and cis-1a-2p are bioactive toward GPR34. In the case of inactive trans-1p-2a, the energy minima (lower than 5.0 kcal/mol) along CV2 were located at around 60° and 150°, and the range between 0° to -180° (CV1) was a high energy region (20.0 kcal/mol) (Figure ). The MTD of cis-1p-2a, which is also inactive toward GPR34 with EC50 >1 µM, showed energy minima along CV2, located around ±60°, while the area from ±180° to ±120° along CV2 is high energy (Figure ). In the case of inactive trans-1p-2a and cis-1p-2a, the bioactive conformations should lie outside the occupied regions, that is, they should lie in the high energy regions. These results confirm that the tetrahydropyran structure restricts the accessible conformations of the molecule due to prohibition of ring flipping and singlebond rotation. While the populations of minor conformers are different between the REMD and MTD calculations, the emergence of conformers is consistent in the two calculations. From the MTD results, the region around CV1 = +60° and CV2 = -180° is consistent with conformations of bioactive cyclic LysoPS derivatives. This region is in agreement with the range of dihedral angles (red box in Table , D1 = +60° and D2 = -180°) obtained by REMD (yellow dashed circle in Figure ). In other words, the conformation involving the coordinates +60° (D1, CV1) and -180° (D2, CV2)) (black dashed circle in Figure ) should be responsible for the bioactivity of LysoPS(18:1) and its cyclic analogues towards GPR34.