Guowei Jiang

Lysobisphosphatidic acid controls endosomal cholesterol levels

Most cell types acquire cholesterol by endocytosis of circulating low density lipoprotein, but little is known about the mechanisms of intra-endosomal cholesterol transport and about the primary cause of its aberrant accumulation in the cholesterol storage disorder Niemann-Pick type C (NPC). Here we report that lysobisphosphatidic acid (LBPA), an unconventional phospholipid that is only detected in late endosomes, regulates endosomal cholesterol levels under the control of Alix/AlP1, which is an LBPA-interacting protein involved in sorting into multivesicular endosomes. We find that Alix down-expression decreases both LBPA levels and the lumenal vesicle content of late endosomes. Cellular cholesterol levels are also decreased, presumably because the storage capacity of endosomes is affected and thus cholesterol clearance accelerated. Both lumenal membranes and cholesterol can be restored in Alix knockdown cells by exogenously added LBPA. Conversely, we also find that LBPA becomes limiting upon pathological cholesterol accumulation in NPC cells, because the addition of exogenous LBPA, but not of LBPA isoforms or analogues, partially reverts the NPC phenotype. We conclude that LBPA controls the cholesterol capacity of endosomes.

α‐Substituted Phosphonate Analogues of Lysophosphatidic Acid (LPA) Selectively Inhibit Production and Action of LPA

Isoform‐selective agonists and antagonists of the lysophosphatidic acid (LPA) G‐protein‐coupled receptors (GPCRs) have important potential applications in cell biology and therapy. LPA GPCRs regulate cancer cell proliferation, invasion, angiogenesis, and biochemical resistance to chemotherapy‐ and radiotherapy‐induced apoptosis. LPA and its analogues are also feedback inhibitors of the enzyme lysophospholipase D (lysoPLD, also known as autotaxin), a central regulator of invasion and metastasis. For cancer therapy, the ideal therapeutic profile would be a metabolically stabilized pan‐LPA receptor antagonist that also inhibits lysoPLD. Herein we describe the synthesis of a series of novel α‐substituted methylene phosphonate analogues of LPA. Each of these analogues contains a hydrolysis‐resistant phosphonate mimic of the labile monophosphate of natural LPA. The pharmacological properties of these phosphono‐LPA analogues were characterized in terms of LPA receptor subtype‐specific agonist and antagonist activity using Ca2+ mobilization assays in RH7777 and CHO cells expressing the individual LPA GPCRs. In particular, the methylene phosphonate LPA analogue is a selective LPA2 agonist, whereas the corresponding α‐hydroxymethylene phosphonate is a selective LPA3 agonist. Most importantly, the α‐bromomethylene and α‐chloromethylene phosphonates show pan‐LPA receptor subtype antagonist activity. The α‐bromomethylene phosphonates are the first reported antagonists for the LPA4 GPCR. Each of the α‐substituted methylene phosphonates inhibits lysoPLD, with the unsubstituted methylene phosphonate showing the most potent inhibition. Finally, unlike many LPA analogues, none of these compounds activate the intracellular LPA receptor PPARγ.

Targeting melanoma growth and viability reveals dualistic functionality of the phosphonothionate analogue of carba cyclic phosphatidic acid

BackgroundAlthough the incidence of melanoma in the U.S. is rising faster than any other cancer, the FDA-approved chemotherapies lack efficacy for advanced disease, which results in poor overall survival. Lysophosphatidic acid (LPA), autotaxin (ATX), the enzyme that produces LPA, and the LPA receptors represent an emerging group of therapeutic targets in cancer, although it is not known which of these is most effective.ResultsHerein we demonstrate that thio-ccPA 18:1, a stabilized phosphonothionate analogue of carba cyclic phosphatidic acid, ATX inhibitor and LPA1/3 receptor antagonist, induced a marked reduction in the viability of B16F10 metastatic melanoma cells compared with PBS-treated control by 80-100%. Exogenous LPA 18:1 or D-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothioate did not reverse the effect of thio-ccPA 18:1. The reduction in viability mediated by thio-ccPA 18:1 was also observed in A375 and MeWo melanoma cell lines, suggesting that the effects are generalizable. Interestingly, siRNA to LPA3 (siLPA3) but not other LPA receptors recapitulated the effects of thio-ccPA 18:1 on viability, suggesting that inhibition of the LPA3 receptor is an important dualistic function of the compound. In addition, siLPA3 reduced proliferation, plasma membrane integrity and altered morphology of A375 cells. Another experimental compound designed to antagonize the LPA1/3 receptors significantly reduced viability in MeWo cells, which predominantly express the LPA3 receptor.ConclusionsThus the ability of thio-ccPA 18:1 to inhibit the LPA3 receptor and ATX are key to its molecular mechanism, particularly in melanoma cells that predominantly express the LPA3 receptor. These observations necessitate further exploration and exploitation of these targets in melanoma.

Aromatic phosphonates inhibit the lysophospholipase D activity of autotaxin

Autotaxin (ATX) is an attractive target for the anticancer therapeutics that inhibits angiogenesis, invasion and migration. ATX is an extracellular lysophospholipase D that hydrolyzes lysophosphatidylcholine to form the bioactive lipid lysophosphatidic acid. The aromatic phosphonate S32826 was the first described nanomolar inhibitor of ATX. However, the tridecylamide substituent on aromatic ring contributed to its poor solubility and bioavailability, severely limiting its utility in vivo. cLogP calculations revealed that the lipophilicity of S32826 could be lowered by shortening its hydrophobic chain and by introducing substituents alpha to the phosphonate. Herein, we describe the synthesis of a small set of α-substituted phosphonate analogs of S32826, and we show that shortening the chain and adding α-halo or α-hydroxy substituents increased solubility; however, ATX inhibition was reduced by most substitutions. An optimal compound was identified for examination of biological effects of ATX inhibition in vivo.

New metabolically stabilized analogues of lysophosphatidic acid: agonists, antagonists and enzyme inhibitors.

Lysophosphatidic acid (LPA) is a metabolically labile natural phospholipid with a bewildering array of physiological effects. We describe herein a variety of long-lived receptor-specific agonists and antagonists for LPA receptors. Several LPA and PA (phosphatidic acid) analogues also inhibit LPP (lipid phosphate phosphatase). The sn-1 or sn-2 hydroxy groups have been replaced by fluorine, difluoromethyl, difluoroethyl, O-methyl or O-hydroxyethoxy groups to give non-migrating LPA analogues that resist acyltransferases. Alkyl ether replacement of acyl esters produced lipase and acyltransferase-resistant analogues. Replacement of the bridging oxygen in the monophosphate by an alpha-monofluoromethylene-, alpha-bromomethylene- or alpha,alpha-difluoromethylenephosphonate gave phosphatase-resistant analogues. Phosphorothioate analogues with O-acyl and O-alkyl chains are potent, long-lived agonists for LPA1 and LPA3 receptors. Most recently, we have (i) prepared stabilized O-alkyl analogues of lysobisphosphatidic acid, (ii) explored the structure-activity relationship of stabilized cyclic LPA analogues and (iii) synthesized neutral head group trifluoromethylsulphonamide analogues of LPA. Through collaborative studies, we have collected data for these stabilized analogues as selective LPA receptor (ant)agonists, LPP inhibitors, TREK (transmembrane calcium channel) K+ channel agonists, activators of the nuclear transcription factor PPAR-gamma (peroxisome-proliferator-activated receptor-gamma), promoters of cell motility and survival, and radioprotectants for human B-cells.

Phosphonothioate and Fluoromethylene Phosphonate Analogues of Cyclic Phosphatidic Acid: Novel Antagonists of Lysophosphatidic Acid Receptors§

Isoform-selective antagonists of the lysophosphatidic acid (LPA) G-protein coupled receptors (GPCRs) have important potential uses in cell biology and clinical applications. Novel phosphonothioate and fluoromethylene phosphonate analogues of carbacyclic phosphatidic acid (ccPA) were prepared by chemical synthesis. The pKa values of these amphilic phosphonolipids and the parent cyclic phosphonate were measured titrimetrically using the Yasuda-Shedlovsky extrapolation. The pharmacological properties of these and other ccPA analogues were characterized for LPA receptor (LPAR) subtype-specific agonist and antagonist activity using Ca2+-mobilization assays in RH7777 cells expressing the individual EDG-family GPCRs. In particular, the phosphonothioate ccPA analogue inhibited Ca2+ release through LPA1/LPA3 activation and was an LPA1/LPA3 antagonist. The monofluoromethylene phosphonate ccPA analogue was also a potent LPA1/LPA3 antagonist. In contrast, the difluoromethylene phosphonate ccPA analogue was a weak LPAR agonist, while ccPA itself had neither agonist nor antagonist activity.

Phosphorothioate analogues of alkyl lysophosphatidic acid as LPA3 receptor-selective agonists

The metabolically stabilized LPA analogue 1‐oleoyl‐2‐O‐methyl‐rac‐glycerophosphorothioate (OMPT) was recently shown to be a potent subtype‐selective agonist for LPA3, a G‐protein‐coupled receptor (GPCR) in the endothelial differentiation gene (EDG) family. Further stabilization was achieved by replacing the sn‐1 O‐acyl group with an O‐alkyl ether. A new synthetic route for the enantiospecific synthesis of the resulting alkyl LPA phosphorothioate analogues is described. The pharmacological properties of the alkyl OMPT analogues were characterized for subtype‐specific agonist activity using Ca2+‐mobilization assays in RH7777 cells expressing the individual EDG family LPA receptors. Alkyl OMPT analogues induced cell migration in cancer cells mediated through LPA1. Alkyl OMPT analogues also activated Ca2+ release through LPA2 activation but with less potency than sn‐1‐oleoyl LPA. In contrast, alkyl OMPT analogues were potent LPA3 agonists. The alkyl OMPTs 1 and 3 induced cell proliferation at submicromolar concentrations in 10T 1/2 fibroblasts. Interestingly, the absolute configuration of the sn‐2 methoxy group of the alkyl OMPT analogues was not recognized by any of the LPA receptors in the EDG family. By using a reporter gene assay for the LPA‐activated nuclear transcription factor PPARγ, we demonstrated that phosphorothioate diesters have agonist activity that is independent of their ligand properties at the LPA‐activated GPCRs. The availability of new alkyl LPA analogues expands the scope of structure–activity studies and will further refine the molecular nature of ligand–receptor interactions for this class of GPCRs.

Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA): metabolically stabilized LPA receptor agonists.

We describe an efficient synthesis of metabolically stabilized sn-2 radyl phosphorothioate analogs of lysophosphatidic acid (LPA), and the determination of the agonist activity of each analog for the six LPA receptors (LPA1-6) using a recently developed TGFα shedding assay. In general, the sn-2 radyl OMPT analogs showed similar agonist activities to the previous 1-oleoyl-2-O-methyl-glycerophosphothioate (sn-1 OMPT) analogs for LPA1-6 receptors. In most cases, the sn-2 radyl-OMPT analogs were more potent agonists than LPA itself. Most importantly, sn-2 alkyl OMPT analogs were very potent LPA5 and LPA6 agonists. The availability of sn-2 radyl OPMT analogs further refines the structure-activity relationships for ligand-receptor interactions for this class of GPCRs.

Bis (monoacylglycero) phosphate interfacial properties and lipolysis by pancreatic lipase-related protein 2, an enzyme present in THP-1 human monocytes.

The interfacial physical properties of bis(monoacylglycero)phosphate (BMP) and its derivatives with three oleoyl chains (hemi-BDP) and four oleoyl chains (bis(diacylglycero)phosphate, BDP) were investigated using Langmuir monomolecular films. The mean molecular area of BMP at the collapse surface pressure (45mN m(-1)) was similar to those measured with other phospholipids bearing two acyl chains (66 and 59.6Å(2) molecule(-1) at pH 5.5 and 8.0, respectively). In Hemi-BDP and BDP, the mean molecular area increased by 26 and 35Å(2) molecule(-1) per additional acyl chain at pH 5.5 and 8.0, respectively. When BMP was added to a phospholipid mixture mimicking late endosome membrane composition at pH 8.0, the mean phospholipid molecular area increased by 7% regardless of the surface pressure. In contrast, the variation in molecular area was surface pressure-dependent at pH 5.5, a pH value close to that of intra-endosomal content. BMP and hemi-BDP, but not BDP, were hydrolyzed by pancreatic lipase-related protein 2 (PLRP2), which exhibits phospholipase A(1) activity. At pH 5.5, the maximum activities of PLRP2 on BMP were recorded at high surface pressures (25-35mN/m). At pH 8.0, the PLRP2 activity vs. surface pressure showed a bell-shaped curve with maximum activities at 15mN/m for both BMP and hemi-BDP. This is a new activity for this enzyme which could degrade cellular BMP since both human PLRP2 (HPLRP2) and BMP were localized in human monocytic THP-1 cells. This is the first report on the cellular localization of HPLRP2 in human monocytes.

Dual Action of Lysophosphatidate-Functionalised Titanium: Interactions with Human (MG63) Osteoblasts and Methicillin Resistant Staphylococcus aureus.

Titanium (Ti) is a widely used material for surgical implants; total joint replacements (TJRs), screws and plates for fixing bones and dental implants are forged from Ti. Whilst Ti integrates well into host tissue approximately 10% of TJRs will fail in the lifetime of the patient through a process known as aseptic loosening. These failures necessitate revision arthroplasties which are more complicated and costly than the initial procedure. Finding ways of enhancing early (osseo)integration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial design. One way of realising improvements in implant quality is to coat the Ti surface with small biological agents known to support human osteoblast formation and maturation at Ti surfaces. Lysophosphatidic acid (LPA) and certain LPA analogues offer potential solutions as Ti coatings in reducing aseptic loosening. Herein we present evidence for the successful bio-functionalisation of Ti using LPA. This modified Ti surface heightened the maturation of human osteoblasts, as supported by increased expression of alkaline phosphatase. These functionalised surfaces also deterred the attachment and growth of Staphylococcus aureus, a bacterium often associated with implant failures through sepsis. Collectively we provide evidence for the fabrication of a dual-action Ti surface finish, a highly desirable feature towards the development of next-generation implantable devices.