The endothelial differentiation gene family encodes three highly homologous G protein-coupled receptors for lysophosphatidic acid (LPA). Based on baculoviral overexpression studies, differences have been proposed in the structure-activity relationship (SAR) of these receptors. We have compared the SAR of the individual receptors either overexpressed transiently at high or at lower levels following stable transfection in LPA-nonresponsive RH7777 cells. The SAR in transfected RH7777 cells was markedly different from that described in insect cells. The LPA 3 receptor has been proposed to be selectively activated by unsaturated LPA species and shows a strong preference for sn-2 versus the sn-1 acyl-LPA regioisomer. Because of the short half-life of sn-2 LPA due to acyl migration under some conditions, we have synthesized acyl migration-resistant analogs using an acetyl group in place of the free hydroxyl group in order to evaluate LPA receptor SAR. Only LPA 1 and LPA 2 showed regioisomeric preference and only for the 18:2 fatty acyl-stabilized LPA sn-1 regioisomer. To identify residues involved in ligand recognition of LPA 3 , we developed and validated computational models of LPA 3 complexes with the analogs studied. The models revealed that Arg-3.28 and Gln-3.29 conserved within the LPA-selective endothelial differentiation gene receptors and the more variable Lys-7.35 and Arg-5.38 of LPA 3 form critical interactions with the polar headgroup of LPA. The models identified Leu-2.60 and Val-7.39 of LPA 3 underlying the regioisomer-selective interaction with the acetyl group of the stabilized regioisomers. Mutation of Leu-2.60 to alanine selectively increased the EC 50 of the sn-2 acetyl-LPA regioisomers, whereas alanine replacement of Val-7.39 profoundly affected both regioisomers.

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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Lipids U 0750Synthesis and Biological Evaluation of Novel Cytotoxic Phospholipids for Prostate Cancer. -Serine amide alcohols and phosphates like (IV), (V), and (VI), derivatives of the natural glycerophospholipid lysophosphatidic acid, represent a new class of cytotoxic phospholipids for prostate cancer. The cytotoxicity is examined in five human prostate cancer cell lines as well as in two negative control cell lines. Compounds (IV), (V), and (VI) are potent inhibitors of prostate tumor cell proliferation at low micromolar cytotoxicity, but they are not selective against nontumor CHO cells and receptor-negative RH777 cells. The chirality of the derivatives is not important for antiproliferative activity. -(GUDUDURU, V.; HURH, E.; DURGAM, G. G.; HONG, S. S.; SARDAR, V. M.; XU, H.; DALTON, J. T.; MILLER*, D. D.; Bioorg. Med.

Public reporting burden for this collection of infonnation is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sourx;es, gathering and maintaining the data needed, and completing and reviewing this collection of infonnation. Send comments regarding this burden estimate or any other aspect of this collection of Infonnation, including suggestions for reducing this burden to Washington Headquarters Services, Directorate for Infonmatlon Operations and Reports, 1215 Jefferson Davis Highway, Suite 1204, Arilr^gton, VA 22202-4302, and to the Office of Management and Budnet,, Washington, DC 20503 AGENCY USE ONLY (Leave blank)2. REPORT DATE April 2003 REPORT TYPE AND DATES COVEREDAnnual Summary (15 Mar 02 -14 Mar 03) TITLE AND SUBTITLEComputational Modeling of the Prostate-Expressed Lysophosphatidic Acid Receptor Edg-76. AUTHOR(S):Vineet M. Sardar, Ph.D. Abby L. Parrill, Ph.D. FUNDING NUMBERSDAMD17-02-1-0248 PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES)The University of Memphis Memphis, Tennessee 38152 E-Mall:vsardar@Memphis.edu Recently it was shown that the lysophosphatidic acid (LPA) receptor LPAs/EDG? is expressed in human prostate cancer cell lines namely LNCap and Du-145. Based on these findings and evidence that LPA is a very potent mitogen, the overall goal of this proposed research is to rationally design LPAs/EDG? selective ligands. We have identified the probable binding position of two LPAi/EDG2 and LPAa/EDG? selective inhibitors namely DGPP (8:0) and FAP (12:0). The binding position and the critical amino acids involved in interacting with these inhibitors relative to the endogenous ligand LPA (18:1) is discussed. PERFORMING ORGANIZATION REPORT NUMBER SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES)U SUBJECT TERMS:Edg-7, computational modeling, acid receptor We explored the binding site and assessed the critical amino acid interactions of antagonists and agonists by docking to the inactive and active form of the receptor respectively.The antagonists, DGPP (8:0) and FAP (12:0), were docked against the inactive receptor models.The agonists, R-LPA (18:1) and S-LPA (18:1) were docked against the active receptor models.One hundred complexes of receptor-ligand pair were generated, in order to evaluate the binding The R and S isomers of LPA (18:1) were docked against the active model of LPA3 to assess the stereo-selectivity of the receptor. A previous study showed that the R and S isomers of 1-C16-GP (Figure 1) were equaUy active in mobilizing Ca^^ in RH7777 cells stably expressing LPAi, LPA2, and LPAa.^^ Based on our results as shovra in Table 1 we do not see any differences in energy with respect to R and S isomer. On assessing the complex of the R isomer of LPA (18:1) with the active form of LPA3 receptor we observe that the polar phosphate ftinctionality is in close proximity to ion-pair with R105 and R276. We also observe that the hydroxyl group at the sn-2 position is in a position to form hydrogen bond with Q106 as shown in figure 4. ...