Lysophosphatidic acid (LPA) is a highly potent endogenous lipid mediator that protects and rescues cells from programmed cell death. Earlier work identified the LPA 2 G proteincoupled receptor subtype as an important molecular target of LPA mediating antiapoptotic signaling. Here we describe the results of a virtual screen using single-reference similarity searching that yielded compounds 2-((9-oxo-9H-fluoren-2-yl) carbamoyl)benzoic acid (NSC12404), 2-((3-(1,3-dioxo-1H-benzo- [de]isoquinolin-2(3H)-yl)propyl)thio)benzoic acid (GRI977143), 4,5-dichloro-2-((9-oxo-9H-fluoren-2-yl)carbamoyl)benzoic acid (H2L55-47924), and 2-((9,10-dioxo-9,10-dihydroanthracen-2-yl)carbamoyl) benzoic acid (H2L5828102), novel nonlipid and drug-like compounds that are specific for the LPA 2 receptor subtype. We characterized the antiapoptotic action of one of these compounds, GRI977143, which was effective in reducing activation of caspases 3, 7, 8, and 9 and inhibited poly(ADP-ribose)polymerase 1 cleavage and DNA fragmentation in different extrinsic and intrinsic models of apoptosis in vitro.Furthermore, GRI977143 promoted carcinoma cell invasion of human umbilical vein endothelial cell monolayers and fibroblast proliferation. The antiapoptotic cellular signaling responses were present selectively in mouse embryonic fibroblast cells derived from LPA 1&2 double-knockout mice reconstituted with the LPA 2 receptor and were absent in vector-transduced control cells. GRI977143 was an effective stimulator of extracellular signal-regulated kinase 1/2 activation and promoted the assembly of a macromolecular signaling complex consisting of LPA 2 , Na ϩ -H ϩ exchange regulatory factor 2, and thyroid receptor interacting protein 6, which has been shown previously to be a required step in LPA-induced antiapoptotic signaling. The present findings indicate that nonlipid LPA 2 -specific agonists represent an excellent starting point for development of lead compounds with potential therapeutic utility for preventing the programmed cell death involved in many types of degenerative and inflammatory diseases.

FTY720 (Fingolimod™), a synthetic analogue of sphingosine 1-phosphate (S1P), activates four of the five EDG-family S1P receptors and is in a phase-III clinical study for the treatment of multiple sclerosis. (S)-FTY720-phosphate (FTY720-P) causes S1P 1 receptor internalization and targeting to the proteasomal degradative pathway, and thus acts as a functional antagonist of S1P 1 by depleting the functional S1P 1 receptor from the plasma membrane. Here we describe the pharmacological characterization of two unsaturated phosphonate enantiomers of FTY720, (R)-and (S)-FTY720-vinylphosphonate. (R)-FTY720-vinylphosphonate was a full agonist of S1P 1 (EC 50 20 ± 3 nM). In contrast, the (S) enantiomer failed to activate any of the five S1P GPCRs and was a full antagonist of S1P 1,3,4 (K i 384 nM, 39 nM, and 1190 nM, respectively) and a partial antagonist of S1P 2 , and S1P 5 . Both enantiomers dose-dependently inhibited lysophospholipase D (recombinant autotaxin) with K i values in the low micromolar range, although with different enzyme kinetic mechanisms. When injected into mice, both enantiomers caused transient peripheral lymphopenia. (R)-and (S)-FTY720-vinylphosphonates activated ERK1/2, AKT, and exerted an antiapoptotic effect in camptothecin-treated IEC-6 intestinal epithelial cells, which primarily express S1P 2 transcripts and traces of S1P 5 . (S)-FTY720-vinylphosphonate is the first pan-antagonist of S1P receptors and offers utility in probing S1P responses in vitro and in vivo. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. The biological effects of the (R)-and (S)-FTY720-vinylphosphonate analogues underscore the complexity of FTY720 cellular targets. NIH Public Access