Serum Autotaxin is not a Useful Biomarker for Ovarian Cancer

Autotaxin (ATX) is a glycoprotein that was first identified in the conditioned medium of human melanoma cells as an autocrine motility factor. It possesses lysophospholipase D activity, producing the bioactive lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. Enhanced expression of ATX mRNA has been reported in various cancer cells and tissues, and it has been speculated that ATX overexpression in cancer cells may be associated with aberrant LPA production. LPA and ATX have been implicated in cancer progression and metastasis, and ovarian cancer is a representative example. In the present study, we measured the serum ATX antigen levels in patients with ovarian cancer and evaluated the usefulness of this parameter for clinical laboratory testing. The serum ATX antigen levels were not increased in ovarian cancer patients as compared with the levels in healthy subjects, and the serum ATX may not be useful as a biomarker for ovarian cancer.     

Autotaxin-LPA-LPP3 Axis in Energy Metabolism and Metabolic Disease

Besides serving as a structural membrane component and intermediate of the glycerolipid metabolism, lysophosphatidic acid (LPA) has a prominent role as a signaling molecule through its binding to LPA receptors at the cell surface. Extracellular LPA is primarily produced from lysophosphatidylcholine (LPC) through the activity of secreted lysophospholipase D, autotaxin (ATX). The degradation of extracellular LPA to monoacylglycerol is mediated by lipid phosphate phosphatases (LPPs) at the cell membrane. This review summarizes and interprets current literature on the role of the ATX-LPA-LPP3 axis in the regulation of energy homeostasis, insulin function, and adiposity at baseline and under conditions of obesity. We also discuss how the ATX-LPA-LPP3 axis influences obesity-related metabolic complications, including insulin resistance, fatty liver disease, and cardiomyopathy.     

Abnormal serum lysophospholipids in multiple myeloma patients

Lysophosphatidylcholine (LPC) and lysophosphatidic acid (LPA) mediate various kinds of biological activities and play an important role in cellular signal transduction. We analyzed serum phospholipids obtained from 16 multiple myeloma (MM) patients and observed that serum LPA level was significantly higher in MM patients (5.3±0.5 nmol/mL) than in normal controls (1.7±0.3 nmol/mL). LPC level was also higher than that in normal controls, and it correlated significantly with the concentration of LPA (r=0.678, P<0.01). In MM patients, palmitic acid/linoleic acid ratios in phosphatidylcholine and LPC were higher than those in normal controls. In the 12-mon follow-up study of two patients with the immune globulin G type, we recognized that the increase of LPC, LPA, and arachidonic acid/linoleic acid ratio in phosphatidylinositol corresponded with a decline in the serum albumin level and choline esterase activity.     

Increased Autotaxin Levels in Severe COVID-19, Correlating with IL-6 Levels,Endothelial Dysfunction Biomarkers,and Impaired Functions of Dendritic Cells

Autotaxin (ATX; ENPP2) is a secreted lysophospholipase D catalyzing the extracellular production of lysophosphatidic acid (LPA), a pleiotropic signaling phospholipid. Genetic and pharmacologic studies have previously established a pathologic role for ATX and LPA signaling in pulmonary injury, inflammation, and fibrosis. Here, increased ENPP2 mRNA levels were detected in immune cells from nasopharyngeal swab samples of COVID-19 patients, and increased ATX serum levels were found in severe COVID-19 patients. ATX serum levels correlated with the corresponding increased serum levels of IL-6 and endothelial damage biomarkers, suggesting an interplay of the ATX/LPA axis with hyperinflammation and the associated vascular dysfunction in COVID-19. Accordingly, dexamethasone (Dex) treatment of mechanically ventilated patients reduced ATX levels, as shown in two independent cohorts, indicating that the therapeutic benefits of Dex include the suppression of ATX. Moreover, large scale analysis of multiple single cell RNA sequencing datasets revealed the expression landscape of ENPP2 in COVID-19 and further suggested a role for ATX in the homeostasis of dendritic cells, which exhibit both numerical and functional deficits in COVID-19. Therefore, ATX has likely a multifunctional role in COVID-19 pathogenesis, suggesting that its pharmacological targeting might represent an additional therapeutic option, both during and after hospitalization.     

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.     

Development of an activity-based probe for autotaxin

Autotaxin (ATX), or ecto-nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2), is a secreted lysophospholipase D that hydrolyses lysophosphatidylcholine into the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX has been implicated in tumour progression and inflammation, and might serve as a biomarker. Here we describe the development of a fluorescent activity-based probe that covalently binds to the active site of ATX. The probe consists of a lysophospholipid-based backbone linked to a trapping moiety that becomes reactive after phosphate ester hydrolysis, and a Cy5 fluorescent dye to allow visualisation of active ATX. The probe reacts specifically with the three known isoforms of ATX, it competes with small-molecule inhibitors for binding to ATX and allows ATX activity in plasma to be determined. Our activity-based reporter will be useful for monitoring ATX activity in biological fluids and for inhibitor screening.     

Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells

Purpose: The objective of the current study was to evaluate the effects of the autotaxin (ATX)–lysophosphatidic acid (LPA) signaling axis on the human trabecular meshwork (HTM) in two-dimensional (2D) and three-dimensional (3D) cultures of HTM cells. Methods: The effects were characterized by transendothelial electrical resistance (TEER) and FITC-dextran permeability (2D), measurements of size and stiffness (3D), and the expression of several genes, including extracellular matrix (ECM) molecules, their modulators, and endoplasmic reticulum (ER) stress-related factors. Results: A one-day exposure to 200 nM LPA induced significant down-sizing effects of the 3D HTM spheroids, and these effects were enhanced slightly on longer exposure. The TEER and FITC-dextran permeability data indicate that LPA induced an increase in the barrier function of the 2D HTM monolayers. A one-day exposure to a 2 mg/L solution of ATX also resulted in a significant decrease in the sizes of the 3D HTM spheroids, and an increase in stiffness was also observed. The gene expression of several ECMs, their regulators and ER-stress related factors by the 3D HTM spheroids were altered by both ATX and LPA, but in different manners. Conclusions: The findings presented herein suggest that ATX may have additional roles in the human TM, in addition to the ATX–LPA signaling axis.     

Hen egg yolk and white contain high amounts of lysophosphatidic acids, growth factor-like lipids: Distinct Molecular species compositions

Hen egg yolk and white were found to contain high amounts of lysophosphatidic acid (acyl LPA) in addition to small amounts of lysoplasmanic acid (alkyl LPA). The levels of acyl LPA in hen egg yolk (44.23 nmol/g tissue) and while (8.81 nmol/g tissue) were on the same order as or higher than the levels of acyl LPA known to be required to elicit biological responses in various animal tissues. Noticeably, there is a marked difference between the fatty acid composition of egg yolk acyl LPA and of egg white acyl LPA; egg yolk acyl LPA predominantly contains saturated fatty acids as the acyl moiety, whereas egg white acyl LPA primarily contains polyunsaturated fatty acids. We found that the level of acyl LPA, especially polyun-saturated fatty acid-containing acyl LPA, in egg white was augmented markedly during the incubation at 37°C, while there was no change in egg yolk. We confirmed that egg white contains both the substrate, i.e., polyunsaturated fatty acid-containing lysophosphatidylcholine (LPC), and the enzyme activity catalyzing the hydrolysis of polyunsaturated fatty acid-containing LPC to the corresponding acyl LPA. Egg yolk LPA and egg white LPA may play separate physiological roles in the development, differentiation, and growth of embryos.     

Enzymatic and chemical synthesis of phosphatidylcholine regioisomers containing eicosapentaenoic acid or docosahexaenoic acid

Regioselective incorporation of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) into phosphatidylcholine (PC) was carried out using enzymatic and chemical synthesis. Incorporation at the sn‐1 position was successfully achieved by lipase‐catalysed esterification of 2‐palmitoyl‐lysophosphatidylcholine (LPC), although in most cases, the enzymes incorporated EPA and DHA at lower rates than other fatty acids. For the incorporation of DHA, Candida antarctica lipase B was the only useful enzyme, while incorporation of EPA was efficiently carried out using either this enzyme or Rhizopus arrhizus lipase. The highest yields in the lipase‐catalysed reactions were obtained at the lowest water activity (close to 0). However, by carrying out the reactions at a higher water activity of 0.22, more EPA and DHA were incorporated. Esterification of 2‐palmitoyl‐LPC with pure EPA at this water activity converted 66 mol‐% of LPC to PC using Rhizopus arrhizus lipase as catalyst. When the fatty acid was DHA and the catalyst Candida antarctica lipase B, 45 mol‐% of PC was obtained. For incorporation of EPA and DHA at the sn‐2 position, phospholipase A₂ was used, but the reaction was very slow. Chemical coupling of 1‐palmitoyl‐LPC and EPA or DHA was more efficient, resulting in complete conversion of LPC.     

Probing Binding and Cellular Activity of Pyrrolidinone and Piperidinone Small Molecules Targeting the Urokinase Receptor

The urokinase receptor (uPAR) is a cell‐surface protein that is part of an intricate web of transient and tight protein interactions that promote cancer cell invasion and metastasis. Here, we evaluate the binding and biological activity of a new class of pyrrolidinone and piperidinone compounds, along with derivatives of previously‐identified pyrazole and propylamine compounds. Competition assays revealed that the compounds displace a fluorescently labeled peptide (AE147‐FAM) with inhibition constant (K_i) values ranging from 6 to 63 μM . Structure‐based computational pharmacophore analysis followed by extensive explicit‐solvent molecular dynamics (MD) simulations and free energy calculations suggested the pyrazole‐based and piperidinone‐based compounds adopt different binding modes, despite their similar two‐dimensional structures. In cells, pyrazole‐based compounds showed significant inhibition of breast adenocarcinoma (MDA‐MB‐231) and pancreatic ductal adenocarcinoma (PDAC) cell proliferation, but piperidinone‐containing compounds exhibited no cytotoxicity even at concentrations of 100 μM . One pyrazole‐based compound impaired MDA‐MB‐231 invasion, adhesion, and migration in a concentration‐dependent manner, while the piperidinone inhibited only invasion. The pyrazole derivative inhibited matrix metalloprotease‐9 (gelatinase) activity in a concentration‐dependent manner, while the piperidinone showed no effect suggesting different mechanisms for inhibition of cell invasion. Signaling studies further highlighted these differences, showing that pyrazole compounds completely inhibited ERK phosphorylation and impaired HIF1α and NF‐κB signaling, while pyrrolidinones and piperidinones had no effect. Annexin V staining suggested that the effect of the pyrazole‐based compound on proliferation was due to cell killing through an apoptotic mechanism. The compounds identified represent valuable leads in the design of further derivatives with higher affinities and potential probes to unravel the protein–protein interactions of uPAR.     

Time‐Dependent Diaryl Ether Inhibitors of InhA: Structure–Activity Relationship Studies of Enzyme Inhibition,Antibacterial Activity,and in vivo Efficacy

The diaryl ethers are a novel class of antituberculosis drug candidates that inhibit InhA, the enoyl‐ACP reductase involved in the fatty acid biosynthesis (FASII) pathway, and have antibacterial activity against both drug‐sensitive and drug‐resistant strains of Mycobacterium tuberculosis. In the present work, we demonstrate that two time‐dependent B‐ring modified diaryl ether InhA inhibitors have antibacterial activity in a mouse model of TB infection when delivered by intraperitoneal injection. We propose that the efficacy of these compounds is related to their residence time on the enzyme, and to identify structural features that modulate drug–target residence time in this system, we have explored the inhibition of InhA by a series of B‐ring modified analogues. Seven ortho‐substituted compounds were found to be time‐dependent inhibitors of InhA, where the slow step leading to the final enzyme–inhibitor complex (EI*) is thought to correlate with closure and ordering of the InhA substrate binding loop. A detailed mechanistic understanding of the molecular basis for residence time in this system will facilitate the development of InhA inhibitors with improved in vivo activity.     

Different Binding Modes of Small and Large Binders of GAT1

Well‐known inhibitors of the γ‐aminobutyric acid (GABA) transporter GAT1 share a common scaffold of a small cyclic amino acid linked by an alkyl chain to a moiety with two aromatic rings. Tiagabine, the only FDA‐approved GAT1 inhibitor, is a typical example. Some small amino acids such as (R)‐nipecotic acid are medium‐to‐strong binders of GAT1, but similar compounds, such as proline, are very weak binders. When substituted with 4,4‐diphenylbut‐3‐en‐1‐yl (DPB) or 4,4‐bis(3‐methylthiophen‐2‐yl)but‐3‐en‐1‐yl (BTB) groups, the resulting compounds have similar pK_i and pIC₅₀ values, even though the pure amino acids have very different values. To investigate if small amino acids and their substituted counterparts share a similar binding mode, we synthesized butyl‐, DPB‐, and BTB‐substituted derivatives of small amino acids. Supported by the results of docking studies, we propose different binding modes not only for unsubstituted und substituted, but also for strong‐ and weak‐binding amino acids. These data lead to the conclusion that following a fragment‐based approach, not pure but N‐butyl‐substituted amino acids should be used as starting points, giving a better estimate of the activity when a BTB or DPB substituent is added.     

Design,Synthesis, and Biological Evaluation of Pyrazoline‐Based Hydroxamic Acid Derivatives as Aminopeptidase N (APN) Inhibitors

Aminopeptidase N (APN) has been recognized as a target for anticancer treatment due to its overexpression on diverse malignant tumor cells and association with cancer invasion, metastasis and angiogenesis. Herein we describe the synthesis, biological evaluation, and structure–activity relationship study of two new series of pyrazoline analogues as APN inhibitors. Among these compounds, 5‐(2‐(2‐(hydroxyamino)‐2‐oxoethoxy)phenyl)‐3‐phenyl‐4,5‐dihydro‐1H‐pyrazole‐1‐carboxamide (compound 13 e ) showed the best APN inhibition with an IC₅₀ value of 0.16±0.02 μm , which is more than one order of magnitude lower than that of bestatin (IC₅₀=9.4±0.5 μm ). Moreover, compound 13 e was found to inhibit the proliferation of diverse carcinoma cells and to show potent anti‐angiogenesis activity. At the same concentration, compound 13 e presents significantly higher anti‐angiogenesis activity than bestatin in human umbilical vein endothelial cells (HUVECs) capillary tube formation assays. The putative binding mode of 13 e in the active site of APN is also discussed.     

Synthesis,Cytotoxicity, Induction of Apoptosis,and Interaction with DNA of Dinuclear Platinum(II) Complexes

Six dicarboxylato‐bridged dinuclear platinum(II) complexes S1 – S6 , with a newly designed chiral ligand, 2‐{[(1R,2R)‐2‐aminocyclohexyl]amino}propanoic acid ( HL ), were prepared and spectrally characterized. The in vitro cytotoxicity of all resulting platinum(II) complexes was evaluated against human HCT‐116, MCF‐7, and HepG‐2 tumor cell lines. The results show that all compounds exhibit positive biological activity toward HCT‐116 and MCF‐7 cell lines, of which complexes S3 , S4 , and S5 , with succinate and its derivatives as bridges, showing better activity than the positive controls. Moreover, double‐dyeing flow cytometric resection experiments indicate that the target compounds inhibit tumor cell growth by inducing apoptosis; gel electrophoresis experiments demonstrate the compounds′ ability to prompt pET22b plasmid DNA degradation in almost the same way as oxaliplatin.     

Stereoselective Modulation of P‐Glycoprotein by Chiral Small Molecules

Inhibition of drug efflux pumps such as P‐glycoprotein (P‐gp) is an approach toward combating multidrug resistance, which is a significant hurdle in current cancer treatments. To address this, N‐substituted aryloxymethyl pyrrolidines were designed and synthesized in their homochiral forms in order to investigate the stereochemical requirements for the binding site of P‐gp. Our study provides evidence that the chiral property of molecules could be a strategy for improving the capacity for interacting with P‐gp, as the most active compounds of the series stereoselectively modulated this efflux pump. The naphthalene‐1‐yl analogue (R)‐2‐[(2,3‐dichlorophenoxy)methyl]‐1‐(naphthalen‐1‐ylmethyl)pyrrolidine) [(R)‐ 7 a ] emerged foremost for its potency and stereoselectivity toward P‐gp, with the S enantiomer being nearly inactive. The modulation of P‐gp by (R)‐ 7 a involved consumption of ATP, thus demonstrating that the compound behaves as a P‐gp substrate.     

Linear and Cyclic Depsipeptidomimetics with β‐Lactam Cores: A Class of New αvβ3 Integrin Receptor Inhibitors

The α_vβ₃ integrin receptor plays an important role in tumor metastasis and tumor‐induced angiogenesis. The inhibition of this receptor with diverse ligands, antibodies, or cyclic peptides is a promising research field for the treatment of a variety of tumors. The replacement of Phe‐(Me)Val dipeptide by a β‐lactam ring in Cilengitide has led to new products that show higher inhibitory activity than the parent cyclopeptide. In particular, substitution of a peptide bond β‐lactam‐NH‐Asp linkage by a β‐lactam‐O‐Asp ester linkage increases the activity of the new cyclodepsipeptide. In the same way it has been found that open‐chain compounds of the form Asp‐β‐lactam‐Arg can interact with the receptor and inhibit its activity moderately. The integrin inhibitory activity of the synthesized compounds has been established by using the CGH array, a method that appears to be a more reliable trial than the classical adhesion test.