Alkoxymethylenephosphonate analogues of (Lyso) phosphatidic acid stimulate signaling networks coupled to the LPA2 receptor

An efficient stereocontrolled synthesis afforded alkoxymethylenephosphonate (MP) analogues of lysophosphatidic acid (LPA) and phosphatidic acid (PA). The pharmacological properties of MP-LPA and MP-PA analogues were characterized for LPA receptor subtype-specific agonist and antagonist activity using Ca(2+)-mobilization assays in RH7777 cells expressing the individual LPA(1)-LPA(3) receptors and CHO cells expressing LPA(4). In addition, activation of a PPARgamma reporter gene construct expressed in CV-1 cells was assessed. These metabolically stabilized LPA analogues exhibited an unexpected pattern of partial agonist/antagonist activity for the LPA G-protein-coupled receptor family and the intracellular LPA receptor PPARgamma. Analogues were compared with 18:1 LPA for activation of downstream signaling in HT-29 colon cancer cells, which exclusively express LPA(2), and both SKOV3 and OVCAR3 ovarian cancer cells, which express LPA(1), LPA(2), and LPA(3). Unexpectedly, reverse phase protein arrays showed that four MP-LPA and MP-PA analogues selectively activated downstream signaling in HT-29 cells with greater potency than LPA. In particular, the oleoyl MP-LPA analogue strongly promoted phosphorylation and activation of AKT, MEK, and pS6 in HT-29 cells in a concentration-dependent manner. In contrast, the four MP-LPA and MP-PA analogues were equipotent with LPA for pathway activation in the SKOV3 and OVCAR3 cells. Taken together, these results suggest that the MP analogues may selectively activate signaling via the LPA(2) receptor subtype, while simultaneously suppressing signaling through the LPA(1) and LPA(3) subtypes.     

The LPA3 Receptor: Regulation and Activation of Signaling Pathways

The lysophosphatidic acid 3 receptor (LPA₃) participates in different physiological actions and in the pathogenesis of many diseases through the activation of different signal pathways. Knowledge of the regulation of the function of the LPA₃ receptor is a crucial element for defining its roles in health and disease. This review describes what is known about the signaling pathways activated in terms of its various actions. Next, we review knowledge on the structure of the LPA₃ receptor, the domains found, and the roles that the latter might play in ligand recognition, signaling, and cellular localization. Currently, there is some information on the action of LPA₃ in different cells and whole organisms, but very little is known about the regulation of its function. Areas in which there is a gap in our knowledge are indicated in order to further stimulate experimental work on this receptor and on other members of the LPA receptor family. We are convinced that knowledge on how this receptor is activated, the signaling pathways employed and how the receptor internalization and desensitization are controlled will help design new therapeutic interventions for treating diseases in which the LPA₃ receptor is implicated.     

G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling

A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)–AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K–AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted.     

Xanthenylacetic Acid Derivatives Effectively Target Lysophosphatidic Acid Receptor 6 to Inhibit Hepatocellular Carcinoma Cell Growth

Despite the increasing incidence of hepatocellular carcinoma (HCC) worldwide, current pharmacological treatments are still unsatisfactory. We have previously shown that lysophosphatidic acid receptor 6 (LPAR6) supports HCC growth and that 9-xanthenylacetic acid (XAA) acts as an LPAR6 antagonist inhibiting HCC growth without toxicity. Here, we synthesized four novel XAA derivatives, (±)-2-(9H-xanthen-9-yl)propanoic acid (compound 4 – MC9), (±)-2-(9H-xanthen-9-yl)butanoic acid (compound 5 – MC6), (±)-2-(9H-xanthen-9-yl)hexanoic acid (compound 7 – MC11), and (±)-2-(9H-xanthen-9-yl)octanoic acid (compound 8 – MC12, sodium salt) by introducing alkyl groups of increasing length at the acetic α-carbon atom. Two of these compounds were characterized by X-ray powder diffraction and quantum mechanical calculations, while molecular docking simulations suggested their enantioselectivity for LPAR6. Biological data showed anti-HCC activity for all XAA derivatives, with the maximum effect observed for MC11. Our findings support the view that increasing the length of the alkyl group improves the inhibitory action of XAA and that enantioselectivity can be exploited for designing novel and more effective XAA-based LPAR6 antagonists.     

Nicotinic Acid Increases Adiponectin Secretion from Differentiated Bovine Preadipocytes through G-Protein Coupled Receptor Signaling

The transition period in dairy cows (3 weeks prepartum until 3 weeks postpartum) is associated with substantial mobilization of energy stores, which is often associated with metabolic diseases. Nicotinic acid (NA) is an antilipolytic and lipid-lowering compound used to treat dyslipidaemia in humans, and it also reduces non-esterified fatty acids in cattle. In mice the G-protein coupled receptor 109A (GPR109A) ligand NA positively affects the secretion of adiponectin, an important modulator of glucose and fat metabolism. In cattle, the corresponding data linking NA to adiponectin are missing. Our objective was to examine the effects of NA on adiponectin and AMPK protein abundance and the expression of mRNAs of related genes such as chemerin, an adipokine that enhances adiponectin secretion in vitro. Differentiated bovine adipocytes were incubated with pertussis toxin (PTX) to verify the involvement of GPR signaling, and treated with 10 or 15 µM NA for 12 or 24 h. NA increased adiponectin concentrations (p ≤ 0.001) and the mRNA abundances of GPR109A (p ≤ 0.05) and chemerin (p ≤ 0.01). Pre-incubation with PTX reduced the adiponectin response to NA (p ≤ 0.001). The NA-stimulated secretion of adiponectin and the mRNA expression of chemerin in the bovine adipocytes were suggestive of GPR signaling-dependent improved insulin sensitivity and/or adipocyte metabolism in dairy cows.     

Epoxyeicosatrienoic Acid and Prostanoid Crosstalk at the Receptor and Intracellular Signaling Levels to Maintain Vascular Tone

Epoxyeicosatrienoic acids (EETs) are signaling lipids produced by the cytochrome P450-(CYP450)-mediated epoxygenation of arachidonic acid. EETs have numerous biological effects on the vascular system, but aspects including their species specificity make their effects on vascular tone controversial. CYP450 enzymes require the 450-reductase (POR) for their activity. We set out to determine the contribution of endothelial CYP450 to murine vascular function using isolated aortic ring preparations from tamoxifen-inducible endothelial cell-specific POR knockout mice (ecPOR^−/−). Constrictor responses to phenylephrine were similar between control (CTR) and ecPOR^−/− mice. Contrastingly, sensitivity to the thromboxane receptor agonist U46619 and prostaglandin E2 (PGE2) was increased following the deletion of POR. Ex vivo incubation with a non-hydrolyzable EET (14,15-EE-8(Z)-E, EEZE) reversed the increased sensitivity to U46619 to the levels of CTR. EETs had no effect on vascular tone in phenylephrine-preconstricted vessels, but dilated vessels contracted with U46619 or PGE2. As U46619 acts through RhoA-dependent kinase, this system was analyzed. The deletion of POR affected the expression of genes in this pathway and the inhibition of Rho-GTPase with SAR407899 decreased sensitivity to U46619. These data suggest that EET and prostanoid crosstalk at the receptor level and that lack of EET production sensitizes vessels to vasoconstriction via the induction of the Rho kinase system.     

alpha,alpha-Cyclopentaneglycine dipeptides capped with biaryls as tachykinin NK(2) receptor antagonists

The NK(2) receptor belongs to the family of tachykinin neurotransmitters. It has been reported to be involved in several pathological conditions, and selective antagonists are potentially useful drugs for the treatment of asthma, irritable bowel syndrome, cystitis, and depression. Starting from in-house capped dipeptide libraries, we were able to identify a number of molecules with sub-nanomolar binding affinity for the hNK(2) receptor. All were characterized by a rigid core structure with a strong constraint induced by an alpha,alpha-cyclopentaneglycine fragment. Herein we report the further elaboration of three initial basic structures. The planar benzothiophene group was substituted with a series of biphenyl and heterobiphenyl moieties that are well tolerated in terms of receptor affinity. The new compounds also maintained good antagonist potency in an in vitro functional assay, and a number of them showed significant in vivo activity after intravenous administration in our guinea pig model.     

Lysophosphatidic Acid Mediates Imiquimod-Induced Psoriasis-like Symptoms by Promoting Keratinocyte Proliferation through LPAR1/ROCK2/PI3K/AKT Signaling Pathway

Psoriasis is a chronic inflammatory skin disease. Recently, lysophosphatidic acid (LPA)/LPAR5 signaling has been reported to be involved in both NLRP3 inflammasome activation in macrophages and keratinocyte activation to produce inflammatory cytokines, contributing to psoriasis pathogenesis. However, the effect and molecular mechanisms of LPA/LPAR signaling in keratinocyte proliferation in psoriasis remain unclear. In this study, we investigated the effects of LPAR1/3 inhibition on imiquimod (IMQ)-induced psoriasis-like mice. Treatment with the LPAR1/3 antagonist, ki16425, alleviated skin symptoms in IMQ-induced psoriasis-like mouse models and decreased keratinocyte proliferation in the lesion. It also decreased LPA-induced cell proliferation and cell cycle progression via increased cyclin A2, cyclin D1, cyclin-dependent kinase (CDK)2, and CDK4 expression and decreased p27^Kip1 expression in HaCaT cells. LPAR1 knockdown in HaCaT cells reduced LPA-induced proliferation, suppressed cyclin A2 and CDK2 expression, and restored p27^Kip1 expression. LPA increased Rho-associated protein kinase 2 (ROCK2) expression and PI3K/AKT activation; moreover, the pharmacological inhibition of ROCK2 and PI3K/AKT signaling suppressed LPA-induced cell cycle progression. In conclusion, we demonstrated that LPAR1/3 antagonist alleviates IMQ-induced psoriasis-like symptoms in mice, and in particular, LPAR1 signaling is involved in cell cycle progression via ROCK2/PI3K/AKT pathways in keratinocytes.     

Alpha-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 alpha-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 Ca(2+) mobilization assays in RH7777 and CHO cells expressing the individual LPA GPCRs. In particular, the methylene phosphonate LPA analogue is a selective LPA(2) agonist, whereas the corresponding alpha-hydroxymethylene phosphonate is a selective LPA(3) agonist. Most importantly, the alpha-bromomethylene and alpha-chloromethylene phosphonates show pan-LPA receptor subtype antagonist activity. The alpha-bromomethylene phosphonates are the first reported antagonists for the LPA(4) GPCR. Each of the alpha-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 PPARgamma.     

Exploring the Active Conformation of Cyclohexane Carboxylate Positive Allosteric Modulators of the Type 4 Metabotropic Glutamate Receptor

The active conformation of a family of metabotropic glutamate receptor subtype 4 (mGlu₄) positive allosteric modulators (PAMs) with the cyclohexane 1,2‐dicarboxylic scaffold present in cis‐2‐(3,5‐dichlorophenylcarbamoyl)cyclohexanecarboxylic acid (VU0155041) was investigated by testing structurally similar six‐membered ring compounds that have a locked conformation. The norbornane and cyclohexane molecules designed as mGlu₄ conformational probes and the enantiomers of the trans diastereomer were computationally characterized and tested in mGlu₄ pharmacological assays. The results support a VU0155041 active conformation, with the chair cyclohexane having the aromatic amide substituent in an axial position and the carboxylate in an equatorial position. Moreover, the receptor displays enantiomeric discrimination of the chiral PAMs. The constructed pharmacophore characterized a highly constrained mGlu₄ allosteric binding site, thus providing a step forward in structure‐based drug design for mGlu₄ PAMs.     

The Versatile 2‐Substituted Imidazoline Nucleus as a Structural Motif of Ligands Directed to the Serotonin 5‐HT1A Receptor

The involvement of the serotonin 5‐HT_1A receptor (5‐HT_1A‐R) in the antidepressant effect of allyphenyline and its analogues indicates that ligands bearing the 2‐substituted imidazoline nucleus as a structural motif interact with 5‐HT_1A‐R. Therefore, we examined the 5‐HT_1A‐R profile of several imidazoline molecules endowed with a common scaffold consisting of an aromatic moiety linked to the 2‐position of an imidazoline nucleus by a biatomic bridge. Our aim was to discover other ligands targeting 5‐HT_1A‐R and to identify the structural features favoring 5‐HT_1A‐R interaction. Structure–activity relationships, supported by modeling studies, suggested that some structural cliché such as a polar function and a methyl group in the bridge, as well as proper steric hindrance in the aromatic area of the above scaffold, favored 5‐HT_1A‐R recognition and activation. We also highlighted the potent antidepressant‐like effect (mouse forced swimming test) of (S)‐(+)‐ 19 [(S)‐(+)‐naphtyline] at very low dose (0.01 mg kg^?1). This effect was clearly mediated by 5‐HT_1A, as it was significantly reduced by pretreatment with the 5‐HT_1A antagonist WAY100635.     

Impact of Substitution Registry on the Receptor-Activation Profiles of Backbone-Modified Glucagon-like Peptide-1 Analogues

Family B G protein-coupled receptors play important physiological roles and possess large extracellular domains (ECDs) that aid in binding the long polypeptide hormones that are their natural agonists. We have previously shown that agonist analogues in which subsets of native α-amino acid residues are replaced with β-amino acid residues can retain activity while avoiding proteolytic degradation. This study focuses on eight new α/β analogues of glucagon-like peptide 1 (GLP-1) that each contain five α-to-β replacements in the C-terminal half of the peptide. This portion of GLP-1 is known to adopt an α-helical conformation and contact the ECD. All four registries of the αααβ backbone pattern were evaluated; previous work has shown that the αααβ pattern supports adoption of an α-helix-like conformation. Two α-to-β replacement formats were employed, one involving β³ homologues of the native residues replaced and the other involving a cyclic β residue. GLP-1R response was characterized in terms of stimulation of cAMP production and β-arrestin recruitment. Some of the backbone-modified GLP-1 analogues display biased agonism of the GLP-1R. This study helps to establish the scope of the α→β backbone modification strategy.     

Thyroid Hormone Signaling in Embryonic Stem Cells: Crosstalk with the Retinoic Acid Pathway

While the role of thyroid hormones (THs) during fetal and postnatal life is well-established, their role at preimplantation and during blastocyst development remains unclear. In this study, we used an embryonic stem cell line isolated from rat (RESC) to study the effects of THs and retinoic acid (RA) on early embryonic development during the pre-implantation stage. The results showed that THs play an important role in the differentiation/maturation processes of cells obtained from embryoid bodies (EB), with thyroid hormone nuclear receptors (TR) (TRα and TRβ), metabolic enzymes (deiodinases 1, 2, 3) and membrane transporters (Monocarboxylate transporters -MCT- 8 and 10) being expressed throughout in vitro differentiation until the Embryoid body (EB) stage. Moreover, thyroid hormone receptor antagonist TR (1-850) impaired RA-induced neuroectodermal lineage specification. This effect was significantly higher when cells were treated with retinoic acid (RA) to induce neuroectodermal lineage, studied through the gene and protein expression of nestin, an undifferentiated progenitor marker from the neuroectoderm lineage, as established by nestin mRNA and protein regulation. These results demonstrate the contribution of the two nuclear receptors, TR and RA, to the process of neuroectoderm maturation of the in vitro model embryonic stem cells obtained from rat.