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.     

Hydrolysis and isomerization of trans,trans-farnesyl diphosphate by Andrographis tissue-culture enzymes.

Incubation of (3R,5S)-[5-3H1]mevalonate + (3RS)-[2-14C]mevalonate with Andrographis cell-free extract leads to trans,trans-farnesol and cis,trans-farnesol which both totally retain tritium. 2. This conflicts with our previous results which predict one third tritium loss in the cis,trans-farnesol. Inversion at C-1 during hydrolysis of trans,trans-farnesyl diphosphate to trans,trans-farnesol could explain this anomaly. 3. (1s)-trans,trans-[1-3H1]Farnesyl diphosphate and phosphate and (1R)-trans,trans-[1-3H1]-farnesyl diphosphate and phosphate, all prepared chemically, were hydrolysed with Andrographis phosphatase, and alkaline phosphatase and hydrogenolysed with lithium aluminium hydride and the product alcohols exchanged with liver alcohol hydrogenase. 4. Both Andrographis phosphatase and alkaline phosphatase hydrolyse trans,trans-farnesyl diphosphate and trans,trans-farnesyl phosphate with retention. 5. Hydrolysis of trans,trans-[1-18O]farnesyl diphosphate in H2(18O with both phosphatases supports P-O fission. 6. The C-1 configuration in (1S)-TRANS,TRANS-[1-3H1]farnesyl diphosphate and phosphate and (1R)-trans,trans-[1-3H1]farnesyl diphosphate and phosphate is progressively racemised in 0.01 M NH4OH/MeOH (1/9) AT - 20 degrees C.     

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.     

Synthesis of acyloxymethyl ester prodrugs of the transferable protein farnesyl transferase substrate farnesyl methylenediphosphonate

Three isoprenoid diphosphate analogues of farnesyl diphosphate (FPP) where the diphosphate has been replaced by methylene diphosphonate and the negative charges masked by frangible pivaloyloxymethyl (POM) esters were prepared. Farnesyl methylenediphosphonate is a sub-micromolar substrate for protein farnesyl transferase. The tripivaloyloxymethyl esters of isoprenoid methylenediphosphonate have significantly increased lipophilicity and may act as important farnesyl diphosphate prodrugs.     

Formation of Z,E,E-geranylgeranyl diphosphate by rat liver microsomes

Rat liver microsomes catalyzed the formation of A,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate in the presence of Triton X-100. Studies on product specificity using various primers such as Z,E-farnesyl diphosphate, E,E-farnesyl diphosphate, Z,E,E-geranylgeranyl diphosphate, E,E,E-geranylgeranyl diphosphate, Z,E,E,E-geranylfarnesyl diphosphate, and E,E,E,E-geranylfarnesyl diphosphate suggested that the microsomal dehydrodolichyl diphosphate synthase has such properties that it releases Z,E,E-geranylgeranyl diphosphate, the first intermediate, in the reactions with farnesyl diphosphate as the starting primer. Metabolic labeling of rat liver slices with [2-3H]mevalonic acid revealed the accumulation of E,E,E-geranylgeranyl (di)phosphates as well as dolichyl (di)phosphate (C85 and C90) and dehydrodolichol (C85 and C90), but no accumulation of Z,E,E-geranylgeranyl (di)phosphate or E,E-farnesyl (di)phosphate was detected. Microsomal enzyme preparations from mouse liver and hamster liver also produced Z,E,E-geranylgeranyl diphosphate from farnesyl diphosphate and isopentenyl diphosphate.     

Initial structure-activity relationships of lysophosphatidic acid receptor antagonists: discovery of a high-affinity LPA1/LPA3 receptor antagonist

A recently reported dual LPA1/LPA3 receptor antagonist (VPC12249, 1) has been modified herein so as to optimize potency and selectivity at LPA receptors. Compounds containing variation in the acyl lipid chain and linker region have been synthesized and screened for activity at individual LPA receptors. LPA1-selective (14b) and LPA3-selective (10g,m) compounds of modest potency have been discovered. Additionally, 2-pyridyl derivative 10t exhibits a Ki value of 18 nM at the LPA1 receptor and is significantly more potent than 1 at the LPA3 receptor. This paper describes the synthetic methods, biological evaluation, and structure-activity relationships (SARs) of LPA receptor antagonists.     

Structural Determinants of the Transient Receptor Potential 1 (TRPV1) Channel Activation by Phospholipid Analogs

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1.     

Activities of Soluble and Microsomal Farnesyl Diphosphatases in Datura stramonium

Farnesyl diphosphatase (FDPase; EC 3.1.7.1) produces farnesol from farnesyl diphosphate (FDP) in a reaction that does not require Mg²⁺. This report shows that FDPase is constitutively expressed at a high level in the soluble and the microsomal fractions of Datura stramonium. Soluble and microsomal FDPase have a similar pH optimum (5.0 – 6.0) and a similar substrate specificity. Geranyl diphosphate (GDP) and geranylgeranyl diphosphate (GGDP) compete for FDP, but isopentenyl diphosphate (IDP), ATP, and para-nitrophenyl phosphate do not. Soluble FDPase activity was highest in fruit and flower followed by root, and leaf. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lysophosphatidic acid inhibits bacterial endotoxin-induced pro-inflammatory response: potential anti-inflammatory signaling pathways

Previous studies have demonstrated that heterotrimeric guanine nucleotide-binding regulatory (Gi) protein-deficient mice exhibit augmented inflammatory responses to lipopolysaccharide (LPS). These findings suggest that Gi protein agonists will suppress LPS-induced inflammatory gene expression. Lysophosphatidic acid (LPA) activates G protein-coupled receptors leading to Gi protein activation. We hypothesized that LPA will inhibit LPS-induced inflammatory responses through activation of Gi-coupled anti-inflammatory signaling pathways. We examined the anti-inflammatory effect of LPA on LPS responses both in vivo and in vitro in CD-1 mice. The mice were injected intravenously with LPA (10 mg/kg) followed by intraperitoneal injection of LPS (75 mg/kg for survival and 25 mg/kg for other studies). LPA significantly increased the mice survival to endotoxemia (P < 0.05). LPA injection reduced LPS-induced plasma TNF-alpha production (69 +/- 6%, P < 0.05) and myeloperoxidase (MPO) activity in lung (33 +/- 9%, P < 0.05) as compared to vehicle injection. LPS-induced plasma IL-6 was unchanged by LPA. In vitro studies with peritoneal macrophages paralleled results from in vivo studies. LPA (1 and 10 microM) significantly inhibited LPS-induced TNFalpha production (61 +/- 9% and 72 +/- 9%, respectively, P < 0.05) but not IL-6. We further demonstrated that the anti-inflammatory effect of LPA was reversed by ERK 1/2 and phosphatase inhibitors, suggesting that ERK 1/2 pathway and serine/threonine phosphatases are involved. Inhibition of phosphatidylinositol 3 (PI3) kinase signaling pathways also partially reversed the LPA anti-inflammatory response. However, LPA did not alter NFkappaB and peroxisome proliferator-activated receptor gamma (PPARgamma) activation. Inhibitors of PPARgamma did not alter LPA-induced inhibition of LPS signaling. These studies demonstrate that LPA has significant anti-inflammatory activities involving activation of ERK 1/2, serine/threonine phosphatases, and PI3 kinase signaling pathways.     

Enzymes encoded by the farnesyl diphosphate synthase gene family in the Big Sagebrush Artemisia tridentata ssp. spiciformis

Farnesyl diphosphate synthase catalyzes the sequential head-to-tail condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate. In plants the presence of farnesyl diphosphate synthase isozymes offers the possibility of differential regulation. Three full-length cDNAs encoding putative isoprenoid synthases, FDS-1, FDS-2, and FDS-5, with greater than 89% similarity were isolated from a Big Sagebrush Artemisia tridentata cDNA library using a three-step polymerase chain reaction protocol. One of the open reading frames, FDS-5, encoded a protein with an N-terminal amino acid extension that was identified as a plastidial targeting peptide. Recombinant histidine-tagged versions of three proteins were purified, and their enzymatic properties were characterized. FDS-1 and FDS-2 synthesized farnesyl diphosphate as the final chain elongation product, but their kinetic behavior varied. FDS-1 prefers geranyl diphosphate over dimethylallyl diphosphate as an allylic substrate and is active at acidic pH values compared with FDS-2. In contrast, FDS-5 synthesized two irregular monoterpenoids, chrysanthemyl diphosphate and lavandulyl diphosphate, when incubated with dimethylallyl diphosphate and an additional product, the regular monoterpene geranyl diphosphate, when incubated with isopentenyl diphosphate and dimethylallyl diphosphate. Specific cellular functions are proposed for each of the three enzymes, and a scenario for evolution of isoprenyl synthases in plants is presented.     

Alendronate is a specific, nanomolar inhibitor of farnesyl diphosphate synthase

Alendronate, a nitrogen-containing bisphosphonate, is a potent inhibitor of bone resorption used for the treatment and prevention of osteoporosis. Recent findings suggest that alendronate and other N-containing bisphosphonates inhibit the isoprenoid biosynthesis pathway and interfere with protein prenylation, as a result of reduced geranylgeranyl diphosphate levels. This study identified farnesyl disphosphate synthase as the mevalonate pathway enzyme inhibited by bisphosphonates. HPLC analysis of products from a liver cytosolic extract narrowed the potential targets for alendronate inhibition (IC(50) = 1700 nM) to isopentenyl diphosphate isomerase and farnesyl diphosphate synthase. Recombinant human farnesyl diphosphate synthase was inhibited by alendronate with an IC(50) of 460 nM (following 15 min preincubation). Alendronate did not inhibit isopentenyl diphosphate isomerase or GGPP synthase, partially purified from liver cytosol. Recombinant farnesyl diphosphate synthase was also inhibited by pamidronate (IC(50) = 500 nM) and risedronate (IC(50) = 3.9 nM), negligibly by etidronate (IC50 = 80 microM), and not at all by clodronate. In osteoclasts, alendronate inhibited the incorporation of [(3)H]mevalonolactone into proteins of 18-25 kDa and into nonsaponifiable lipids, including sterols. These findings (i) identify farnesyl diphosphate synthase as the selective target of alendronate in the mevalonate pathway, (ii) show that this enzyme is inhibited by other N-containing bisphosphonates, such as risendronate, but not by clodronate, supporting a different mechanism of action for different bisphosphonates, and (iii) document in purified osteoclasts alendronate inhibition of prenylation and sterol biosynthesis.     

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.     

Kinetic studies of Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase reaction using 3-desmethyl allylic substrate analogs

In order to investigate the substrate binding feature of undecaprenyl diphosphate synthase from Micrococcus luteus B-P 26 with respect to farnesyl diphosphate and a reaction intermediate, (Z,E,E)-geranylgeranyl diphosphate, we examined the reactivity of artificial substrate analogs, 3-desmethyl farnesyl diphosphate and 3-desmethyl Z-geranylgeranyl diphosphate, which lack the methyl group at the 3-position of farnesyl diphosphate and Z-geranylgeranyl diphosphate, respectively. Undecaprenyl diphosphate synthase did not accept either of the 3-desmethyl analogs as the allylic substrate, indicating that the methyl group at the 3-position of the allylic substrate is important in the undecaprenyl diphosphate synthase reaction. These analogs showed different inhibition patterns in the cis-prenyl chain elongation reaction with respect to the reactions of farnesyl diphosphate and Z-geranylgeranyl diphosphate as allylic substrate. These results suggest that the binding site for the natural substrate farnesyl diphosphate and those for the intermediate allylic diphosphate, which contains the cis-prenyl unit, are different during the cis-prenyl chain elongation reaction.     

Farnesyl diphosphate synthase is a cytosolic enzyme in Leishmania major promastigotes and its overexpression confers resistance to risedronate

Farnesyl diphosphate synthase is the most likely molecular target of aminobisphosphonates (e.g., risedronate), a set of compounds that have been shown to have antiprotozoal activity both in vitro and in vivo. This protein, together with other enzymes involved in isoprenoid biosynthesis, is an attractive drug target, yet little is known about the compartmentalization of the biosynthetic pathway. Here we show the intracellular localization of the enzyme in wild-type Leishmania major promastigote cells and in transfectants overexpressing farnesyl diphosphate synthase by using purified antibodies generated towards a homogenous recombinant Leishmania major farnesyl diphosphate synthase protein. Indirect immunofluorescence, together with immunoelectron microscopy, indicated that the enzyme is mainly located in the cytoplasm of both wild-type cells and transfectants. Digitonin titration experiments also confirmed this observation. Hence, while the initial step of isoprenoid biosynthesis catalyzed by 3-hydroxy-3-methylglutaryl-coenzyme A reductase is located in the mitochondrion, synthesis of farnesyl diphosphate by farnesyl diphosphate synthase is a cytosolic process. Leishmania major promastigote transfectants overexpressing farnesyl diphosphate synthase were highly resistant to risedronate, and the degree of resistance correlated with the increase in enzyme activity. Likewise, when resistance was induced by stepwise selection with the drug, the resulting resistant promastigotes exhibited increased levels of farnesyl diphosphate synthase. The overproduction of protein under different conditions of exposure to risedronate further supports the hypothesis that this enzyme is the main target of aminobisphosphonates in Leishmania cells.     

Sphingosine 1-phosphate, lysophosphatidic acid and growth factor signaling and termination

Sphingosine 1 phosphate (S1P) and lysophosphatidic acid (LPA) are bioactive lipid phosphates that bind to cell surface G-protein coupled receptors (GPCR) and, in addition, exhibit intracellular actions. We have summarised herein, an important functional interaction between lipid phosphate GPCR and receptor tyrosine kinases (RTK) that enables growth factors to spatially regulate effectors, thereby governing the nature of the biological response. For instance, we describe how the formation of functional complexes between the S1P(1) receptor and PDGFbeta receptor may effectively re-programme platelet-derived growth factor from a mitogenic to a migratory stimulus. This is achieved by integration of RTK- and GPCR-specific signals that results in spatial regulation of a cytoplasmic retained pool of extracellular signal regulated kinase-1/2 linked to myosin light chain kinase, myosin light chain phosphorylation and migration. We therefore suggest that the lipid phosphate receptor is a major determinant in regulating growth factor-dependent biology. Growth factors can also increase S1P inside cells, and we discuss the concept of spatial/temporal aspects of compartmentalised intracellular signaling of S1P in relation to defined interactions between, for instance, sphingosine kinase, phospholipase D1 and lipid phosphate phosphatases and regulation of cell survival.