Despite an intriguing cell biology and the suggestion of a role in pathophysiological responses, the mechanism of action of such lipid phosphoric acid mediators as lysophosphatidic acid (LPA) remains obscure, in part because of an underdeveloped medicinal chemistry. We report now the agonist activity of a synthetic phospholipid in which the glycerol backbone of LPA is replaced by L-serine. Like LPA, the L-serine-based lipid mobilizes calcium and inhibits activation of adenylyl cyclase in the human breast cancer cell line MDA MB231. Treatment with LPA desensitizes MDA MB231 cells to subsequent application of the L-serine compound; when the order of application is reversed, however, the L-serine compound does not prevent calcium mobilization by LPA, which might indicate the existence of two LPA receptors in these cells. The analogous D-serine-based phospholipid was distinctly less potent than the L-isomer in those assays; this finding demonstrates stereoselectivity by an LPA receptor. Unlike LPA, the L-serine-based lipid does not evoke a chloride conductance in Xenopus laevis oocytes, but injection of poly(A) ϩ RNA from HEK 293 cells confers this phenotype on the oocyte. The latter result has practical importance in that it allows use of the frog oocyte for expression cloning of an LPA receptor DNA, an assay system made problematic by the oocyte's strong endogenous response to LPA.

Although lipid phosphoric acid mediators such as lysophosphatidic acid (LPA) are now recognized widely as intercellular signaling molecules, the medicinal chemistry of these mediators is poorly developed. With the goal of achieving a better understanding of the structure activity relationships in LPA, we have synthesized and tested a series of LPA analogs that lack the 2-hydroxyl moiety. Our series consisted of compounds with 2, 3, or 4 carbon diol or amino alcohol backbones and oleoyl or palmitoleoyl acyl groups. These molecules cannot be acylated further to form phosphatidic acids, nor do they have chiral centers. The rank order potency of these compounds in mobilization of calcium in MDA MB-231 cells suggested a maximum optimal chain length of 24-25 atoms. However, high potency for the inhibition of adenylyl cyclase in these cells was achieved only by one compound that also contained a dissociable proton five bond lengths from the phosphorus atom. That compound, N-oleoyl-2-hydroxyethyl-1-phosphate, was nearly equipotent to 1-oleoyl LPA in both assays. The striking mimicry of LPA by the ethanolamine-based compound and the presence of fatty acid amides in tissue prompts us to propose that phosphorylated N-acyl ethanolamides occur naturally.

Summary:Purpose: Previously we proposed and provided evidence for the metabolic pathway of felbamate (FBM), which leads to the reactive metabolite, 3-carbamoyl-2-phenylpropionaldehyde. This aldehyde carbamate was suggested to be the reactive intermediate in the oxidation of 2-phenyl-1,3-propanediol monocarbamate to the major human metabolite 3-carbamoyl-2-phenylpropionic acid. In addition, the aldehyde carbamate was found to undergo spontaneous elimination to 2-phenylpropenal, commonly known as atropaldehyde. Moreover, atropaldehyde was proposed to play a role in the development of toxicity during FBM therapy. Evidence for atropaldehyde formation in vivo was reported with the identification of modified N-acetyl-cysteine conjugates of atropaldehyde in both human and rat urine after FBM administration. Identification of the atropaldehyde-derived mercapturic acids in urine after FBM administration is consistent with the hypothesis that atropaldehyde is formed in vivo and that it reacts with thiol nucleophiles. Based on the hypothesis that the potential for toxicity will correlate to the amount of atropaldehyde formed, we sought to develop an analytic method that would quantify the amount of relevant metabolites excreted in patient urine.Methods: We summarize the results of an LClMS method used to quantify FBM, 3-carbamoyl-2-phenylpropionic acid and two atropaldehyde-derived mercapturic acids in the patient population.Results: Analysis was performed on 3 I patients undergoing FBM therapy. The absolute quantities of FBM and three metabolites were measured.Conclusions: This method demonstrated sufficient precision for the identification of patients exhibiting "abnormal" levels of atropaldehyde conjugates and may hold potential for patient monitoring.

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Lysophosphatidic acid (LPA) has associated with it an intriguing cell biology that is thought to be mediated through its interaction with G-protein coupled receptor(s). In an effort to extend the structure-activity relationships of LPA, we have produced a series of LPA analogues in which the glycerol core in LPA was replaced with conformationally restricted aryl substructures. The aryl substructures encompassed aminophenol, resorcinol, dihydroxy benzophenone, and tocopherol systems. The benzophenone moiety was investigated both as a conformationally restricting substructure for LPA and as a possible photoreactive alkylating agent for the LPA receptor(s). All LPA analogues were evaluated for their potency and efficacy in mobilizing calcium ions from internal stores in MDA MB-231 cells. Ten of the 14 analogues exhibited activity in this assay at doses up to 5 microM; none of the compounds exhibited nonreceptor-mediated lytic activity at this maximal concentration. The receptor response showed surprising tolerance for manipulation in the backbone region of LPA, although none of the compounds were equipotent to LPA. This tolerance for a variety of structures has given us new leads into the realization of novel agonists and antagonists of the LPA receptor(s).

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