Synthesis and pharmacological evaluation of second-generation phosphatidic acid derivatives as lysophosphatidic acid receptor ligands

Short-chain phosphatidic acid derivatives, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 1) and phosphatidic acid 8:0 (PA 8:0, 2), were previously identified as subtype-selective LPA(1) and LPA(3) receptor antagonists. Recently, we reported that the replacement of the phosphate headgroup by thiophosphate in a series of fatty alcohol phosphates (FAP) improves agonist as well as antagonist activities at LPA GPCR. Here, we report the synthesis of stereoisomers of PA 8:0 analogs and their biological evaluation at LPA GPCR, PPARgamma, and ATX. The results indicate that LPA receptors stereoselectively interact with glycerol backbone modified ligands. We observed entirely stereospecific responses by dioctyl PA 8:0 compounds, in which (R)-isomers were found to be agonists and (S)-isomers were antagonists of LPA GPCR. From this series, we identified compound 13b as the most potent LPA(3) receptor subtype-selective agonist (EC(50)=3 nM), and 8b as a potent and selective LPA(3) receptor antagonist (K(i)=5 nM) and inhibitor of ATX (IC(50)=600 nM). Serinediamide phosphate 19b was identified as an LPA(3) receptor specific antagonist with no effect on LPA(1), LPA(2), and PPARgamma.     

Structure-activity relationships of lysophosphatidic acid analogs

The physiologic effects of lysophosphatidic acid (LPA) remain poorly understood. Our ignorance is due in part to lack of medicinal chemistry focussed on this pleiotropic lipid mediator. Beginning with commercially available phospholipids tested on whole cells or tissues and continuing with synthetic analogs tested at recombinant LPA receptors, the features of the LPA pharmacophore have become visible. An active LPA mimetic has a long aliphatic chain terminating in a phosphate monoester; bulky substitutions at the second carbon (relative to the phosphate) are tolerated poorly and a dissociable proton near the phosphate group seems required for optimal activity. These requirements are met by substituting ethanolamine for the glyceryl group in LPA. Substitutions at the second carbon of the N-acyl ethanolamide phosphoric acid (NAEPA) result in highly active agonists, including some receptor type selective compounds, if the substituent is small (e.g. methyl, methylene amino, methylene hydroxy). However, bulky hydrophobic substituents lead to compounds with decreased agonist, or even antagonist, activities. Examination of naturally occurring plant lipids led to the discovery of another LPA receptor antagonist, di-octyl glyceryl pyrophosphate. An unexplained result obtained in testing the LPA mimetics is the strong stereoselectivity exhibited by some responses (e.g. calcium mobilization) and the lack of stereoselectivity of other responses (e.g. platelet aggregation).     

Molecular basis for lysophosphatidic acid receptor antagonist selectivity

Recent characterization of lysophosphatidic acid (LPA) receptors has made possible studies elucidating the structure-activity relationships (SAR) for agonist activity at individual receptors. Additionally, the availability of these receptors has allowed the identification of antagonists of LPA-induced effects. Two receptor-subtype selective LPA receptor antagonists, one selective for the LPA1/EDG2 receptor (a benzyl-4-oxybenzyl N-acyl ethanolamide phosphate, NAEPA, derivative) and the other selective for the LPA3/EDG7 receptor (diacylglycerol pyrophosphate, DGPP, 8:0), have recently been reported. The receptor SAR for both agonists and antagonists are reviewed, and the molecular basis for the difference between agonism and antagonism as well as for receptor-subtype antagonist selectivity identified by molecular modeling is described. The implications of the newly available receptor-subtype selective antagonists are also discussed.     

Alkyl lysophosphatidic acid and fluoromethylene phosphonate analogs as metabolically-stabilized agonists for LPA receptors

We describe an efficient method for the synthesis of alkyl lysophosphatidic acid (LPA) analogs as well as alkyl LPA mono- and difluoromethylene phosphonate analogs. Each alkyl LPA analog was evaluated for subtype-specific LPA receptor agonist activity using a cell migration assay for LPA(1) activation in cancer cells and an intracellular calcium mobilization assay for LPA(2) and LPA(3) activation. Alkyl LPAs induced pronounced cell migration activity with equivalent or higher potency than sn-1-oleoyl LPA, while the alkyl LPA fluoromethylene phosphonates proved to be less potent agonists in this assay. However, each alkyl LPA analog activated Ca(2+) release by activation of LPA(2) and LPA(3) receptors. Interestingly, the absolute configuration of the sn-2 hydroxyl group of the alkyl LPA analogs was not recognized by any of the three LPA receptors. The use of alkyl LPA analogs further expands the scope of structure-activity studies, which will better define LPA-LPA receptor interactions.     

Inhibition of transcellular tumor cell migration and metastasis by novel carba-derivatives of cyclic phosphatidic acid

Cyclic phosphatidic acid (1-acyl-sn-glycerol-2,3-cyclic phosphate; cPA) is a naturally occurring analog of lysophosphatidic acid (LPA) with a variety of distinctly different biological activities from those of LPA. In contrast to LPA, a potent inducer of tumor cell invasion, palmitoyl-cPA inhibits FBS- and LPA-induced transcellular migration and metastasis. To prevent the conversion of cPA to LPA we synthesized cPA derivatives by stabilizing the cyclic phosphate ring; to prevent the cleavage of the fatty acid we generated alkyl ether analogs of cPA. Both sets of compounds were tested for inhibitory activity on transcellular tumor cell migration. Carba derivatives, in which the phosphate oxygen was replaced with a methylene group at either the sn-2 or the sn-3 position, showed much more potent inhibitory effects on MM1 tumor cell transcellular migration and the pulmonary metastasis of B16-F0 melanoma than the natural pal-cPA. The antimetastatic effect of carba-cPA was accompanied by the inhibition of RhoA activation and was not due to inhibition of the activation of LPA receptors.     

Mechanism of rapid elimination of lysophosphatidic acid and related lipids from the circulation of mice

Lysophosphatidic acid (LPA) is a bioactive lipid mediator. Concentrations of the major LPA species in mouse plasma decreased uniformly following administration of a potent selective inhibitor of the LPA-generating lysophospholipase D autotaxin, identifying an active mechanism for removal of LPA from the circulation. LPA, akylglycerol phosphate (AGP), sphingosine 1-phosphate (S1P), and a variety of structural mimetics of these lipids, including phosphatase-resistant phosphonate analogs of LPA, were rapidly eliminated (t1/2 < 30 s) from the circulation of mice following intravenous administration of a single bolus dose without significant metabolism in situ in the blood. These lipids accumulated in the liver. Elimination of intravenously administered LPA was blunted by ligation of the hepatic circulation, and ∼90% of LPA administered through the portal vein was accumulated by the isolated perfused mouse liver at first pass. At early times following intravenous administration, more LPA was associated with a nonparenchymal liver cell fraction than with hepatocytes. Primary cultures of nonparenchymal liver cells rapidly assimilated exogenously provided LPA. Our results identify hepatic uptake as an important determinant of the bioavailability of LPA and bioactive lysophospholipid mimetics and suggest a mechanism to explain changes in circulating LPA levels that have been associated with liver dysfunction in humans.     

Thrombogenic and atherogenic activities of lysophosphatidic acid

Lysophosphatidic acid (LPA) has been identified as a biologically active lipid in mildly-oxidized LDL, human atherosclerotic lesions, and the supernatant of activated platelets. The evidence that LPA has thrombogenic and atherogenic activities has increased substantially in recent years. Supporting the thrombogenic activity of LPA, analysis of the core region of human carotid plaques revealed recently the presence of alkyl- and acyl-molecular species from LPA with high platelet-activating potency (16:0 alkyl-LPA, 20:4 acyl-LPA). LPA, lipid extracts of atherosclerotic plaques, and the lipid-rich core elicited shape change and, in synergy with other platelet stimuli, aggregation of isolated platelets. This effect was completely abrogated by prior incubation of platelets with LPA receptor antagonists. Furthermore, LPA at concentrations approaching those found in vivo, induced platelet shape change, aggregation, and platelet-monocyte aggregate formation in blood. LPA-stimulated platelet aggregation was mediated by the ADP-stimulated activation of the P2Y(1) and P2Y(12) receptors. Supporting its atherogenic activity, LPA is a mitogen and motogen to vascular smooth muscle cells (VSMCs) and an activator of endothelial cells and macrophages. Recently, LPA has been identified as an agonist of the peroxisome proliferator activating receptor gamma (PPARgamma), which is a key regulator of atherogenesis. LPA elicits progressive neointima formation, which is fully abolished by GW9662, an antagonist of PPARgamma. We propose that LPA plays a central role in eliciting vascular remodeling and atherogenesis. Furthermore, upon rupture of lipid-rich atherosclerotic plaques, LPA may trigger platelet aggregation and intra-arterial thrombus formation. Antagonists of LPA receptors might be useful in preventing LPA-elicited thrombus formation and neointima formation in patients with cardiovascular diseases.     

Identification of Darmstoff analogs as selective agonists and antagonists of lysophosphatidic acid receptors

Darmstoff describes a family of gut smooth muscle-stimulating acetal phosphatidic acids initially isolated and characterized from the bath fluid of stimulated gut over 50 years ago. Despite similar structural and biological profiles, Darmstoff analogs have not previously been examined as potential LPA mimetics. Here, we report a facile method for the synthesis of potassium salts of Darmstoff analogs. To understand the effect of stereochemistry on lysophosphatidic acid mimetic activity, synthesis of optically pure stereoisomers of selected Darmstoff analogs was achieved starting with chiral methyl glycerates. Each Darmstoff analog was evaluated for subtype-specific LPA receptor agonist/antagonist activity, PPARgamma activation, and autotaxin inhibition. From this study we identified compound 12 as a pan-antagonist and several pan-agonists for the LPA(1-3) receptors. Introduction of an aromatic ring in the lipid chain such as analog 22 produced a subtype-specific LPA(3) agonist with an EC(50) of 692 nM. Interestingly, regardless of their LPA(1/2/3) ligand properties all of the Darmstoff analogs tested activated PPARgamma. However, these compounds are weak inhibitors of autotaxin. The results indicate that Darmstoff analogs constitute a novel class of lysophosphatidic acid mimetics.     

Stereochemical properties of lysophosphatidic acid receptor activation and metabolism

Ligand recognition by G protein-coupled receptors (GPCR), as well as substrate recognition by enzymes, almost always shows a preference for a naturally occurring enantiomer over the unnatural one. Recognition of lysophosphatidic acid (LPA) by its receptors is an exception, as both the natural L (R) and unnatural D (S) stereoisomers of LPA are equally active in bioassays. In contrast to the enantiomers of LPA, analogs of N-acyl-serine phosphoric acid (NASPA) and N-acyl-ethanolamine phosphoric acid (NAEPA), which contain a serine and an ethanolamine backbone, respectively, in place of glycerol, are recognized in a stereoselective manner. This stereoselective interaction may lead to the development of receptor subtype-selective antagonists. In the present study, we review the stereochemical aspects of LPA pharmacology and describe the chemical synthesis of pure LPA enantiomers together with their ligand-binding properties toward the LPA1, LPA2, and LPA3 receptors and their metabolism by lipid phosphate phosphatase 1 (LPP1). Finally, we evaluate the concept of stereopharmacology in developing novel ligands for LPA receptors.     

The role of lysophosphatidic acid receptors in phenotypic modulation of vascular smooth muscle cells

Lysophosphatidic acid (LPA) is a bioactive lipid with diverse physiological effects via activation of G protein-coupled receptors (GPCRs). It has been implicated as a specific dedifferentiation factors that can promote phenotypic modulation of cultured vascular smooth muscle cells (VSMCs) which is critically involved in various vascular disease. However, the role of LPA receptors and details of their signaling in LPA induced phenotypic modulation are largely unexplored. In this study we detect the expression of LPA1 and LPA3 in rat aortic smooth muscle cells (RASMCs). LPA promoted RASMCs phenotypic modulation in a dose-dependent manner and coordinated induced the phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and extracellular signal-regulated kinase (ERK). LPA-induced cell phenotypic modulation was significantly inhibited by specific LPA1/LPA3-receptor antagonist dioctyl-glycerol pyrophosphate (DGPP8:0) at concentration, but this inhibitive effect was lost when the antagonist was coadministered with a highly selective LPA3 agonist,1-oleoyl-2-Omethyl-rac-glycero-phosphothionate (OMPT). In addition, pertussis toxin (PTX), a Gi protein inhibitor had little affect on the LPA-induced phenotypic modulation in RASMC. These data suggest that LPA-induced phenotypic modulation is mediated through the PTX-insensitive G-protein(s), possibly Gq-coupled LPA3 receptor.     

Quantitative analysis of lysophosphatidic acid by time-of-flight mass spectrometry using a phosphate-capture molecule

Lysophosphatidic acid (LPA) is a lipid mediator that may play an important role in wound healing, embryonic development, and progression of cancer. Here, we report a procedure for the quantification of LPA by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The method is based on a characteristic mass shift with total charge change (from -2 to +1) of the phosphate species due to 1:1 complexation of LPA(2-) with a dinuclear zinc (II) complex [1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato dizinc(II) complex; Zn(2)L(3+)] at physiological pH. The monocationic complex [LPA(2-)-Zn(2)L(3+)](+) was detected in the positive mode, in which no other signal of cation adducts of LPA(2-) was observed. The detection limit of 18:1 LPA by this method was 0.1 pmol on a sample plate. The intensity ratio of [LPA(2-)-Zn(2)L(3+)](+) against an internal standard [17:0 LPA(2-)-Zn(2)L(3+)](+) increased linearly with their molar ratio. Based on the relative intensities of complex ions, we determined the amounts of LPA homologs in an egg white by this method; the results obtained were in good agreement with those by gas liquid chromatography. This sensitive and convenient procedure for LPA-specific detection is useful for the quantification of LPA homologs occurring in biological materials.     

Cyclic phosphatidic acid and lysophosphatidic acid induce hyaluronic acid synthesis via CREB transcription factor regulation in human skin fibroblasts

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator and an analog of the growth factor-like phospholipid lysophosphatidic acid (LPA). cPA has a unique cyclic phosphate ring at the sn-2 and sn-3 positions of its glycerol backbone. We showed before that a metabolically stabilized cPA derivative, 2-carba-cPA, relieved osteoarthritis pathogenesis in vivo and induced hyaluronic acid synthesis in human osteoarthritis synoviocytes in vitro. This study focused on hyaluronic acid synthesis in human fibroblasts, which retain moisture and maintain health in the dermis. We investigated the effects of cPA and LPA on hyaluronic acid synthesis in human fibroblasts (NB1RGB cells). Using particle exclusion and enzyme-linked immunosorbent assays, we found that both cPA and LPA dose-dependently induced hyaluronic acid synthesis. We revealed that the expression of hyaluronan synthase 2 messenger RNA and protein is up-regulated by cPA and LPA treatment time dependently. We then characterized the signaling pathways up-regulating hyaluronic acid synthesis mediated by cPA and LPA in NB1RGB cells. Pharmacological inhibition and reporter gene assays revealed that the activation of the LPA receptor LPAR₁, G_i/o protein, phosphatidylinositol-3 kinase (PI3K), extracellular-signal-regulated kinase (ERK), and cyclic adenosine monophosphate response element-binding protein (CREB) but not nuclear factor κB induced hyaluronic acid synthesis by the treatment with cPA and LPA in NB1RGB cells. These results demonstrate for the first time that cPA and LPA induce hyaluronic acid synthesis in human skin fibroblasts mainly through the activation of LPAR₁-G_i/o followed by the PI3K, ERK, and CREB signaling pathway.     

The critical micelle concentrations of lysophosphatidic acid and sphingosylphosphorylcholine

The critical micelle concentrations (CMC) of lysophosphatidic acid (LPA) and sphingosylphosphorylcholine (SPC) were measured by isothermal titration calorimetry. The CMC of LPA decreases with salt concentration and acyl chain length. In water at 25 °C, the CMC values of 1-acyl-2-lyso-sn-glycero-3-phosphatidic acid are 1.850, 0.540, 0.082, and 0.346 mM, respectively, when the acyl group is myristoyl, palmitoyl, stearoyl, and oleoyl. The CMC of SPC in 10 mM sodium phosphate buffer, pH 7.4, at 25 °C was 0.158 mM, and did not change with an increase in salt concentration.     

Specific LPA receptor subtype mediation of LPA-induced hypertrophy of cardiac myocytes and involvement of Akt and NFkappaB signal pathways

Lysophosphatidic acid (LPA) is a bioactive phospholipid with diverse functions mediated via G-protein-coupled receptors (GPCRs). In view of the elevated levels of LPA in acute myocardial infarction (MI) patients we have conducted studies aimed at identifying specific LPA receptor subtypes and signaling events that may mediate its actions in hypertrophic remodeling. Experiments were carried out in cultured neonatal rat cardiomyocytes (NRCMs) exposed to LPA and in a rat MI model. In NRCMs, LPA-induced hypertrophic growth was completely abrogated by DGPP, an LPA1/LPA3 antagonist. The LPA3 agonist OMPT, but not the LPA2 agonist dodecylphosphate, promoted hypertrophy as examined by 3[H]-Leucine incorporation, ANF-luciferase expression and cell area. In in vivo experiments, LPA1, LPA2 and LPA3 mRNA levels as well as LPA1 and LPA3 protein levels increased together with left ventricular remodeling (LVRM) after MI. In addition, LPA stimulated the phosphorylation of Akt and p65 protein and activated NF-kappaB-luciferase expression. Inhibitors of PI3K (wortmannin), mTOR (rapamycin), and NF-kappaB (PDTC or SN50) effectively prevented LPA-induced 3[H]-Leucine incorporation and ANF-luciferase expression. Furthermore, ERK inhibitors (U0126 and PD98059) suppressed LPA-stimulated activation of NF-kappaB and p65 phosphorylation whereas wortmannin showed no effect on NF-kappaB activation. Our findings indicate that LPA3 and/or LPA1 mediate LPA-induced hypertrophy of NRCMs and that LPA1 and LPA3 may be involved in LVRM of MI rats. Moreover, Akt and NF-kappaB signaling pathways independently implicate in LPA-stimulated myocardial hypertrophic growth.     

A novel series of 2-pyridyl-containing compounds as lysophosphatidic acid receptor antagonists: development of a nonhydrolyzable LPA3 receptor-selective antagonist

A recently reported dual LPA(1)/LPA(3) receptor antagonist (1) has been modified so as to modulate the basicity, sterics, and dipole moment of the 2-pyridyl moiety. Additionally, the implications of installing nonhydrolyzable phosphate head group isosteres with regard to antagonist potency and selectivity at LPA receptors is described. This study has resulted in the development of the first nonhydrolyzable and presumably phosphatase-resistant LPA(3)-selective antagonist reported to date.     

Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity

Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production.     

Identification of a phosphothionate analogue of lysophosphatidic acid (LPA) as a selective agonist of the LPA3 receptor

Lysophosphatidic acid (LPA) is a bioactive lysophospholipid mediator that acts through G protein-coupled receptors. Most cell lines in culture express one or more LPA receptors, making it difficult to assign a response to specific LPA receptors. Dissection of the signaling properties of LPA has been hampered by lack of LPA receptor subtype-specific agonists and antagonists. The present study characterizes an ester-linked thiophosphate derivative (1-oleoyl-2-O-methyl-rac-glycerophosphothionate, OMPT) of LPA. OMPT is a functional LPA analogue with potent mitogenic activity in fibroblasts. In contrast to LPA, OMPT does not couple to the pheromone response through the LPA(1) receptor in yeast cells. OMPT induces intracellular calcium increases efficiently in LPA(3) receptor-expressing Sf9 cells but poorly in LPA(2) receptor-expressing cells. Guanosine 5'-O-(3-[(35)S]thio)triphosphate binding assays in mammalian cells showed that LPA exhibits agonistic activity on all three LPA receptor subtypes, whereas OMPT has a potent agonistic effect only on the LPA(3) receptor. In transiently transfected HEK293 cells, OMPT stimulates mitogen-activated protein kinases through the LPA(3) but not the LPA(1) or LPA(2) receptors. Furthermore, OMPT-induced intracellular calcium mobilization in mammalian cells is efficiently inhibited by the LPA(1)/LPA(3) receptor-selective antagonist VPC12249. These results establish that OMPT is an LPA(3)-selective agonist. OMPT binding to the LPA(3) receptor in mammalian cells is sufficient to elicit multiple responses, including activation of G proteins, calcium mobilization, and activation of mitogen-activated protein kinases. Thus OMPT offers a powerful probe for the dissection of LPA signaling events in complex mammalian systems.     

Subtype-specific residues involved in ligand activation of the endothelial differentiation gene family lysophosphatidic acid receptors

Lysophosphatidic acid (LPA) is a ligand for three endothelial differentiation gene family G protein-coupled receptors, LPA(1-3). We performed computational modeling-guided mutagenesis of conserved residues in transmembrane domains 3, 4, 5, and 7 of LPA(1-3) predicted to interact with the glycerophosphate motif of LPA C18:1. The mutants were expressed in RH7777 cells, and the efficacy (E(max)) and potency (EC(50)) of LPA-elicited Ca(2+) transients were measured. Mutation to alanine of R3.28 universally decreased both the efficacy and potency in LPA(1-3) and eliminated strong ionic interactions in the modeled LPA complexes. The alanine mutation at Q3.29 decreased modeled interactions and activation in LPA(1) and LPA(2) more than in LPA(3). The mutation W4.64A had no effect on activation and modeled LPA interaction of LPA(1) and LPA(2) but reduced the activation and modeled interactions of LPA(3). The R5.38A mutant of LPA(2) and R5.38N mutant of LPA(3) showed diminished activation by LPA; however, in LPA(1) the D5.38A mutation did not, and mutation to arginine enhanced receptor activation. In LPA(2), K7.36A decreased the potency of LPA; in LPA(1) this same mutation increased the E(max). In LPA(3), R7.36A had almost no effect on receptor activation; however, the mutation K7.35A increased the EC(50) in response to LPA 10-fold. In LPA(1-3), the mutation Q3.29E caused a modest increase in EC(50) in response to LPA but caused the LPA receptors to become more responsive to sphingosine 1-phosphate (S1P). Surprisingly micromolar concentrations of S1P activated the wild type LPA(2) and LPA(3) receptors, indicating that S1P may function as a weak agonist of endothelial differentiation gene family LPA receptors.