However, there has been opposition to the induced conformational fit mechanism based on the idea that the "biologically active" conformation may be too unfavorable in the unbound state and may only appear as a result of ligand-protein interactions. In general, the conformation of a bound ligand should be stabilized as compared with the unbound ligand; in other words, the interaction energy between the ligand and the binding site (e.g., stabilization by the formation of hydrogen bonds) should be greater than the energy associated with the transition from the unbound to the bound state. Bioactive small molecules (e.g., drugs) usually have a limited number of rotatable bonds, suggesting that a marked dissimilarity between the non-binding and bioactive binding states would be highly unfavorable. However, some highly active endogenous ligands, including lipidic ligands, have a large number of rotatable bonds, and in these cases the occurrence of a bioactive conformation in the conformational ensemble of unbound states seems energetically plausible. In other words, the "bioactive" conformation will always be present in the unbound state, even if the fraction of this conformation is small. Thus, although some authors have argued that adopting a bioactive conformation represents a large energy penalty for a drug, others support the idea that the bioactive conformation is an intrinsic component of the unbound ligand, so that binding involves little energy penalty. The design of efficient ligands always requires a compromise between rigidity, which lowers the entropic cost of binding, and flexibility, which increases the likelihood of favorable interactions between ligand and protein. In this context, conformational restrictions such as cyclization have frequently been used as a strategy to modify the bioactivity of ligands. We considered that this approach could be used to identify a ligand's bioactive conformational space by comparing the bioactivity and accessible conformational space of a series of structurally restricted analogs. To test this idea, we focused here on the endogenous lipidic ligand lysophosphatidylserine (LysoPS) (Figure ). Lysophosphatidylserine (LysoPS) is generated by the enzymatic deacylation of phosphatidylserine (PS). It is an endogenous ligand of GPCRs, and specifically recognizes the three human GPCRs, LPS1 (GPR34), LPS2 (P2Y10) and LPS3 (GPR174). These receptors have roles in various biological functions, including activation/inactivation of the immune system in pathological settings. This panagonistic activity of LysoPS raises the intriguing question of whether the bioactive conformations of LysoPS binding to these receptors are different. LysoPS can be regarded as consisting of three distinct modules (fatty acid, glycerol, and L-serine) connected by phosphodiester and ester linkages. We previously optimized the structure of each module and the ester linkage of the endogenous ligand (1-oleoyl-LysoPS, 1, LysoPS(18:1)), and obtained potent and receptor subtype-selective modulators of GPR34, P2Y10 and GPR174. (MD) also supported the putative GPR34-LysoPS(18:1) docking pose. In addition, we showed that conformational constraint of the glycerol part of LysoPS structure altered the activity toward GPR34 (Figure and Table ). Our present study further confirms that the agonistic activity of LysoPS can be modulated by structural modification of the glycerol part (Table ). These experimental facts are consistent with the idea that LysoPS has a different bioactive binding pose for each of these receptors.