We previously reported that fatty alcohol phosphates (FAP) represent a minimal pharmacophore required to interact with lysophosphatidic acid (LPA) receptors. To improve the activity of the first-generation saturated FAP series, a structure−activity relationship (SAR) study was carried out that includes modifications to the headgroup and alkyl side chain of the FAP pharmacophore. A series of unsaturated (C10−C18) FAP, headgroup-modified hydrolytically stable saturated (C10−C18) alkyl phosphonates, and saturated and unsaturated (C10−C18) thiophosphate analogues were synthesized and evaluated for activity in RH7777 cells transfected with individual LPA1 - 3 receptors, in PC-3 cells and in human platelets that endogenously express all three isoforms. In this series we identified several LPA1- and LPA3-selective antagonists with IC50 values in the nanomolar range. Oleoyl-thiophosphate (15g) was shown to be a pan-agonist, whereas tetradecyl-phosphonate (16c) was identified as a pan-antagonist. These compounds were also tested for the ability to activate the transcription factor PPARγ, an intracellular receptor for LPA, in CV1 cells transfected with the PPRE-Acox-Rluc reporter gene. All the FAP tested, along with the previously reported LPA GPCR antagonists dioctanoyl glycerol pyrophosphate (2), Ki16425 (6), and the agonist OMPT (3), were activators of PPARγ. The pan-agonist oleoyl-thiophosphate (15g) and pan-antagonist tetradecyl-phosphonate (16c) mimicked LPA in inhibiting autotaxin, a secreted lysophospholipase D that produces LPA in biological fluids.

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P 1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head groupinteracting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/ L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [ 32 P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P 1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P 1 .Sphingosine 1-phosphate (S1P)3 (see Fig. 1) is a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type (1-3). S1P plays fundamental physiological roles in vascular stabilization (4), heart development (5), lymphocyte homing (6), and cancer angiogenesis (7). S1P elicits its biological effects through the activation of G protein-coupled receptors (GPCR) (8 -10) and through yet undefined intracellular targets (11-15). The endothelial differentiation gene (EDG) family of GPCR encodes eight highly homologous receptors. Five of these receptors, designated S1P 1 -S1P 5 , are specific for S1P, and the other three, LPA 1 -LPA 3 , are specific for the related lysophospholipid mediator lysophosphatidic acid (LPA) (16).FTY-720, an immunosuppressive prodrug presently in phase 3 clinical trials, has attracted a lot of interest due to its effective inhibition of kidney transplant rejection and attenuation of autoimmune diseases, including multiple sclerosis (6,17,18). In vivo, FTY-720 becomes phosphorylated by sphingosine kinase type 2, and FTY-720-P is a high affinity ligand of all EDG family S1P receptors with the exception of S1P 2 (19). In atrial myocytes, FTY-720-P, similarly to S1P (20, 21), activates an inwardly rectifying K ϩ conductance through the activation of the S1P 3 receptor, which in turn elicits unwanted bradyca...

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The dissociation constant for an ionizable ligand binding to a receptor is dependent on its charge and therefore on its environmentally-influenced pK a value. The pK a values of sphingosine 1-phosphate (S1P) were studied computationally in the context of the wild type S1P 1 receptor and the following mutants: E3.29Q, E3.29A, and K5.38A. Calculated pK a values indicate that S1P binds to S1P 1 and its site mutants with a total charge of −1, including a +1 charge on the ammonium group and a −2 charge on the phosphate group. The dissociation constant of S1P binding to these receptors was studied as well. The models of wild type and mutant proteins originated from an active receptor model that was developed previously. We used ab initio RHF/6-31+G(d) to optimize our models in aqueous solution, where the solvation energy derivatives are represented by conductor-like polarizable continuum model (C-PCM) and integral equation formalism polarizable continuum model (IEF-PCM). Calculation of the dissociation constant for each mutant was determined by reference to the experimental dissociation constant of the wild type receptor. The computed dissociation constants of the E3.29Q and E3.29A mutants are 3-5 orders of magnitude higher than those for the wild type receptor and K5.38A mutant, indicating vital contacts between the S1P phosphate group and the carboxylate group of E3.29. Computational dissociation constants for K5.38A, E3.29A and E3.29Q mutants were compared with experimentally determined binding and activation data. No measurable binding of S1P to the E3.29A and E3.29Q mutants was observed, supporting the critical contacts observed computationally. These results validate the quantitative accuracy of the model.

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