Background & Aims-We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. The objective of the present study was to evaluate the effect of octadecenyl thiophosphate (OTP), a novel rationally designed, metabolically stabilized LPA mimic, on radiation-induced apoptosis of intestinal epithelial cells in vitro and in vivo

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.

A more complete understanding of the physiological and pathological role of lysophosphatidic acid (LPA) requires receptor subtype-specific agonists and antagonists. Here, we report the synthesis and pharmacological characterization of fatty alcohol phosphates (FAP) containing saturated hydrocarbon chains from 4 to 22 carbons in length. Selection of FAP as the lead structure was based on computational modeling as a minimal structure that satisfies the two-point pharmacophore developed earlier for the interaction of LPA with its receptors. Decyl and dodecyl FAPs (FAP-10 and FAP-12) were specific agonists of LPA 2 (EC 50 ϭ 3.7 Ϯ 0.2 M and 700 Ϯ 22 nM, respectively), yet selective antagonists of LPA 3 (K i ϭ 90 nM for FAP-12) and FAP-12 was a weak antagonist of LPA 1 . Neither LPA 1 nor LPA 3 receptors were activated by FAPs; in contrast, LPA 2 was activated by FAPs with carbon chains between 10 and 14. Computational modeling was used to evaluate the interaction between individual FAPs (8 to 18) with LPA 2 by docking each compound in the LPA binding site. FAP-12 displayed the lowest docked energy, consistent with its lower observed EC 50 . The inhibitory effect of FAP showed a strong hydrocarbon chain length dependence with C12 being optimum in the Xenopus laevis oocytes and in LPA 3 -expressing RH7777 cells. FAP-12 did not activate or interfere with several other G-protein-coupled receptors, including S1P-induced responses through S1P 1,2,3,5 receptors. These data suggest that FAPs are ligands of LPA receptors and that FAP-10 and FAP-12 are the first receptor subtype-specific agonists for LPA 2 .

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Lysophosphatidic acid (LPA), a component of mildly-oxidized LDL and the lipid rich core of atherosclerotic plaques, elicits platelet activation. LPA is the ligand of G protein-coupled receptors (GPCR) of the EDG family (LPA 1-3 ) and the newly identified LPA 4-7 subcluster. LPA 4 , LPA 5 and LPA 7 increase cellular cAMP levels that would induce platelet inhibition rather than activation. In the present study we quantified the mRNA levels of the LPA 1-7 GPCR in human platelets and found a rank order LPA 4 =LPA 5 >LPA 7 >LPA 6 =LPA 2 >>LPA 1 >LPA 3 . We examined platelet shape change using a panel of LPA receptor subtype-selective agonists and antagonists and compared them with their pharmacological profiles obtained in heterologous LPA 1-5 receptor expression systems. Responses to different natural acyl and alkyl species of LPA, and octyl phosphatidic acid analogs, alpha-substituted phosphonate analogs, N-palmitoyl-tyrosine phosphoric acid, N-palmitoyl-serine phosphoric acid were tested. All of these compounds elicited platelet activation and also inhibited LPA-induced platelet shape change after pre-incubation, suggesting that receptor desensitization is likely responsible for the inhibition of this response. Fatty acid free albumin (10 µM) lacking platelet activity completely inhibited platelet shape change induced by LPA with an IC 50 of 1.1 µM but had no effect on the activation of LPA 1,2,3,&5 expressed in endogenously non-LPA-responsive RH7777 cells. However, albumin reduced LPA 4 activation and shifted the dose-response curve to the right. LPA 5 transiently expressed in RH7777 cells showed preference to alkyl-LPA over acyl-LPA that is similar to that in platelets. LPA did not increase cAMP levels in platelets. In conclusion, our results with the pharmacological compounds and albumin demonstrate that LPA does not induce platelet shape change simply through activation of LPA 1-5, and the receptor(s) mediating LPA-induced platelet activation remains elusive.