Identification of two novel chemical classes of Autotaxin (ATX) inhibitors using Enalos Asclepios KNIME nodes.

Autotaxin is a secreted lysophospholipase D which is a member of the ectonucleotide pyrophosphatase/phosphodiesterase family converting extracellular lysophosphatidylcholine and other non-choline lysophospholipids, such as lysophosphatidylethanolamine and lysophosphatidylserine, to the lipid mediator lysophosphatidic acid. Autotaxin is implicated in various fibroproliferative diseases including interstitial lung diseases, such as idiopathic pulmonary fibrosis and hepatic fibrosis, as well as in cancer. In this study, we present an effort of identifying ATX inhibitors that bind to allosteric ATX binding sites using the Enalos Asclepios KNIME Node. All the available PDB crystal structures of ATX were collected, prepared, and aligned. Visual examination of these structures led to the identification of four crystal structures of human ATX co-crystallized with four known inhibitors. These inhibitors bind to five binding sites with five different binding modes. These five binding sites were thereafter used to virtually screen a compound library of 14,000 compounds to identify molecules that bind to allosteric sites. Based on the binding mode and interactions, the docking score, and the frequency that a compound comes up as a top-ranked among the five binding sites, 24 compounds were selected for in vitro testing. Finally, two compounds emerged with inhibitory activity against ATX in the low micromolar range, while their mode of inhibition and binding pattern were also studied. The two derivatives identified herein can serve as "hits" towards developing novel classes of ATX allosteric inhibitors.

Isosteric Replacement of Ester Linkage of Lysophospholipids with Heteroaromatic Rings Retains Potency and Subtype Selectivity.

Our group has reported various derivatives of lysophosphatidylserine (LysoPS) as potent and subtype-selective agonists for G-protein-coupled receptors (GPCRs). However, the ester linkage between the glycerol moiety and fatty acid or fatty acid surrogate is present in all of them. In order to develop these LysoPS analogs as drug candidates, appropriate pharmacokinetic consideration is essential. Here, we found that the ester bond of LysoPS is highly susceptible to metabolic degradation in mouse blood. Accordingly, we examined isosteric replacement of the ester linkage with heteroaromatic rings. The resulting compounds showed excellent retention of potency and receptor subtype selectivity, as well as increased metabolic stability in vitro.

Autotaxin facilitates selective LPA receptor signaling.

Autotaxin (ATX; ENPP2) produces the lipid mediator lysophosphatidic acid (LPA) that signals through disparate EDG (LPA1-3) and P2Y (LPA4-6) G protein-coupled receptors. ATX/LPA promotes several (patho)physiological processes, including in pulmonary fibrosis, thus serving as an attractive drug target. However, it remains unclear if clinical outcome depends on how different types of ATX inhibitors modulate the ATX/LPA signaling axis. Here, we show that the ATX "tunnel" is crucial for conferring key aspects of ATX/LPA signaling and dictates cellular responses independent of ATX catalytic activity, with a preference for activation of P2Y LPA receptors. The efficacy of the ATX/LPA signaling responses are abrogated more efficiently by tunnel-binding inhibitors, such as ziritaxestat (GLPG1690), compared with inhibitors that exclusively target the active site, as shown in primary lung fibroblasts and a murine model of radiation-induced pulmonary fibrosis. Our results uncover a receptor-selective signaling mechanism for ATX, implying clinical benefit for tunnel-targeting ATX inhibitors.

Influenza Virus Membrane Fusion Is Promoted by the Endosome-Resident Phospholipid Bis(monoacylglycero)phosphate.

The phospholipid bis(monoacylglycero)phosphate (BMP) is enriched in late endosomal and endolysosomal membranes and is believed to be involved in membrane deformation and generation of intralumenal vesicles within late endosomes. Previous studies have demonstrated that BMP promotes membrane fusion of several enveloped viruses, but a limited effect has been found on influenza virus. Here, we report the use of single-virus fusion assays to dissect BMP's effect on influenza virus fusion in greater depth. In agreement with prior reports, we found that hemifusion kinetics and efficiency were unaffected by the addition of 10-20 mol % BMP to the target membrane. However, using an assay for fusion pore formation and genome exposure, we found full fusion efficiency to be substantially enhanced by the addition of 10-20 mol % BMP to the target membrane, while the kinetics remained unaffected. By comparing BMP to other negatively charged phospholipids, we found the effect on fusion efficiency mainly attributable to headgroup charge, although we also hypothesize a role for BMP's unusual chemical structure. Our results suggest that BMP function as a permissive factor for a wider range of viruses than previously reported. We hypothesize that BMP may be a general cofactor for endosomal entry of enveloped viruses.

Syntheses of Symmetrical and Unsymmetrical Lysobisphosphatidic Acid Derivatives.

Recent years have witnessed significant achievements in the field of organic chemistry, which have led to new drugs and the discovery of new and biologically interesting molecules. Herein, we describe a practical and efficient approach to the synthesis of enantiomerically pure and diverse lysobisphosphatidic acid analogues. The key feature of the synthesis is a one-pot, sequential phosphorylation of a protected sn-2-O-oleoyl glycerol or sn-3-O-oleoyl glycerol with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite, followed by oxidation.

Persistent elevation of lysophosphatidylcholine promotes radiation brain necrosis with microglial recruitment by P2RX4 activation.

Brain radiation necrosis (RN) or neurocognitive disorder is a severe adverse effect that may occur after radiation therapy for malignant brain tumors or head and neck cancers. RN accompanies inflammation which causes edema or micro-bleeding, and no fundamental treatment has been developed. In inflammation, lysophospholipids (LPLs) are produced by phospholipase A2 and function as bioactive lipids involved in sterile inflammation in atherosclerosis or brain disorders. To elucidate its underlying mechanisms, we investigated the possible associations between lysophospholipids (LPLs) and RN development in terms of microglial activation with the purinergic receptor P2X purinoceptor 4 (P2RX4). We previously developed a mouse model of RN and in this study, measured phospholipids and LPLs in the brains of RN model by liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses. We immune-stained microglia and the P2RX4 in the brains of RN model with time-course. We treated RN model mice with ivermectin, an allosteric modulator of P2RX4 and investigate the effect on microglial activation with P2RX4 and LPLs' production, and resulting effects on overall survival and working memory. We revealed that LPLs (lysophosphatidylcholine (LPC), lysophosphatidyl acid, lysophosphatidylserine, lysophosphatidylethanolamine, lysophosphatidylinositol, and lysophosphatidylglycerol) remained at high levels during the progression of RN with microglial accumulation, though phospholipids elevations were limited. Both microglial accumulation and activation of the P2RX4 were attenuated by ivermectin. Moreover, the elevation of all LPLs except LPC was also attenuated by ivermectin. However, there was limited prolongation of survival time and improvement of working memory disorders. Our findings suggest that uncontrollable increased LPC, even with ivermectin treatment, promoted the development of RN and working memory disorders. Therefore, LPC suppression will be essential for controlling RN and neurocognitive disorder after radiation therapy.

Biophysical Characterization of Tolerogenic Lipid-Based Nanoparticles Containing Phosphatidylcholine and Lysophosphatidylserine.

Autoimmune conditions, allergies, and immunogenicity against therapeutic proteins are initiated by the unwanted immune response against self and non-self proteins. The development of tolerance induction approaches can offer an effective treatment modality for these clinical conditions. We recently showed that oral administration of lipidic nanoparticles containing phosphatidylcholine (PC) and lysophosphatidylserine (Lyso-PS) converted an immunogen to a tolerogen and induced immunological tolerance towards several antigens. While the biophysical properties such as lamellar characteristics of this binary lipid system are critical for stability, therapeutic delivery, and mechanism of tolerance induction, such information has not been thoroughly investigated. In the current study, we evaluated the lamellar phase properties of PC/Lyso-PS system using orthogonal biophysical methods such as fluorescence (steady-state, anisotropy, PSvue, and Laurdan), dynamic light scattering, and differential scanning calorimetry. The results showed that Lyso-PS partitioned into the PC bilayers and led to changes in the particles' lamellar phase properties, lipid-packing, and lipid-water dynamics. Additionally, the biophysical characteristics of PC/Lyso-PS system are different from the well-studied PC/double-chain phosphatidylserine (PS) system. Notably, the incorporation of Lyso-PS significantly reduced the hydrodynamic diameter of PC particles. Results from the in vivo uptake study and intestinal loop assay utilizing flow cytometry analysis also indicated that the uptake of Lyso-PS-containing nanoparticles by immune cells in the gut and Peyer's patches is significantly higher than that of double-chain PS due to the differential transport through microfold cells. It was also found that the acyl chain mismatch between PC and Lyso-PS is critical for the miscibility and particle stability. Collectively, the results suggest that these biophysical characteristics likely influence the in vivo behaviors and contribute to the oral tolerance property of PC/Lyso-PS system.

Characterization of recombinant murine GDE4 and GDE7, enzymes producing lysophosphatidic acid and/or cyclic phosphatidic acid.

GDE4 and GDE7 are membrane-bound enzymes that exhibit lysophospholipase D activities. We found that GDE7 produced not only lysophosphatidic acid (LPA) but also cyclic phosphatidic acid (cPA) from lysophospholipids by a transphosphatidylation reaction. In contrast, GDE4 produced only LPA. The analysis of substrate specificity showed that 1-alkyl-lysophosphospholipids were preferred substrates for both enzymes rather than 1-alkyl-lysophospholipids and 1-alkenyl-lysophospholipids. Among the various lysophospholipids with different polar head groups that were tested, lysophosphatidylglycerol and lysophosphatidylserine were preferred substrates for GDE4 and GDE7, respectively. The detailed analysis of the dependency of the enzyme activities of GDE4 and GDE7 on divalent cations suggested multiple divalent cations were bound in the active sites of both enzymes. Taken together, these results suggest the possibility that GDE7 functions as a cPA-producing enzyme in the body.

18:0 Lyso PC, a natural product with potential PPAR-γ agonistic activity, plays hypoglycemic effect with lower liver toxicity and cardiotoxicity in db/db mice.

Rosiglitazone, a specific agonist of peroxisome proliferator-activated receptor-γ (PPAR-γ), displays a robust hypoglycemic action in patients with type 2 diabetes mellitus (T2DM) and elicits serious adverse reactions, especially hepatotoxicity and cardiotoxicity. Here, we aims to find a new natural PPAR-γ agonist with less adverse reactions than rosiglitazone in db/db mice. The method of virtual screening was used to identify a PPAR-γ agonist 18:0 Lyso PC from an in-house natural product library. We verified its pharmacological effects and adverse reactions comparing with rosiglitazone in vivo and in vitro. 18:0 Lyso PC exhibited pharmacological effects similar to those of rosiglitazone in db/db mice. Moreover, 18:0 Lyso PC showed a lower extent of liver injury and cardiotoxicity in db/db mice. The mechanism, by which this natural compound alleviates metabolic syndrome, involves a reduction in fatty acid synthesis mediated by activation of the phosphorylation of adenosine 5'-monophosphate (AMP)-activated protein kinase-alpha (AMPKα) and acetyl-CoA carboxylase (ACC) and an increase expression of uncoupled protein 1 (UCP1) and PPAR-γ coactivator-1 alpha (PGC1-α). 18:0 Lyso PC, a natural compound, can show a similar hypoglycemic effect to rosiglitazone by activating PPAR-γ, while eliciting markedly fewer adverse reactions than rosiglitazone.

Structural basis of the P4B ATPase lipid flippase activity.

P4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic α-subunit and accessory ß-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a ß-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1's ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.

2-Carba-lysophosphatidic acid is a novel ß-lysophosphatidic acid analogue with high potential for lysophosphatidic acid receptor activation and autotaxin inhibition.

Cyclic phosphatidic acid (cPA) is a naturally occurring phospholipid mediator that, along with its chemically stabilized analogue 2-carba-cyclic phosphatidic acid (2ccPA), induces various biological activities in vitro and in vivo. Although cPA is similar to lysophosphatidic acid (LPA) in structure and synthetic pathway, some of cPA biological functions apparently differ from those reported for LPA. We previously investigated the pharmacokinetic profile of 2ccPA, which was found to be rapidly degraded, especially in acidic conditions, yielding an unidentified compound. Thus, not only cPA but also its degradation compound may contribute to the biological activity of cPA, at least for 2ccPA. In this study, we determined the structure and examined the biological activities of 2-carba-lysophosphatidic acid (2carbaLPA) as a 2ccPA degradation compound, which is a type of ß-LPA analogue. Similar to LPA and cPA, 2carbaLPA induced the phosphorylation of the extracellular signal-regulated kinase and showed potent agonism for all known LPA receptors (LPA1-6) in the transforming growth factor-α (TGFα) shedding assay, in particular for LPA3 and LPA4. 2carbaLPA inhibited the lysophospholipase D activity of autotaxin (ATX) in vitro similar to other cPA analogues, such as 2ccPA, 3-carba-cPA, and 3-carba-LPA (α-LPA analogue). Our study shows that 2carbaLPA is a novel ß-LPA analogue with high potential for the activation of some LPA receptors and ATX inhibition.

Establishment of a novel, cell-based autotaxin assay.

Autotaxin (ATX) plays an important role in (patho-)physiological lysophosphatidic acid (LPA) signaling. Here we describe the establishment of novel cell-based ATX assay formats. ATX-mediated LPA generation is detected by using a stable LPA receptor reporter cell line. In a first assay variant, ATX-mediated LPA generation is started in the absence of cells and the reaction mix is transferred to the reporter cells after stopping the reaction (two-tube assay). In a second assay variant, ATX is added to the reporter cells expressing the known autotaxin binding partners integrin ß1, integrin ß3 and the LPA receptor 1. LPA generation is started in the presence of cells and is detected in real-time (one-tube assay). Structurally diverse ATX inhibitors with different binding modes were characterized in both cell-based assay variants and were also tested in the well-established biochemical choline release assay. ATX inhibitors displayed similar potencies, regardless if the assay was performed in the absence or presence of cells, and comparable results were obtained in all three assay formats. In summary, our novel cell-based ATX assay formats are well-suited for sensitive detection of enzyme activity as well as for the characterization of ATX inhibitors in the presence and absence of cells.

Pharmacological and structure-activity relationship studies of oleoyl-lysophosphatidylinositol synthetic mimetics.

Metabolic diseases, such as obesity and type 2 diabetes, are relentlessly spreading worldwide. The beginning of the 21st century has seen the introduction of mechanistically novel types of drugs, aimed primarily at keeping these pathologies under control. In particular, an important family of therapeutics exploits the beneficial physiology of the gut-derived glucagon-like peptide-1 (GLP-1), with important clinical benefits, from glycaemic control to cardioprotection. Nonetheless, these protein-based drugs act systemically as exogenous GLP-1 mimetics and are not exempt from side effects. The food-derived lipid oleoyl-lysophosphatidylinositol (LPI) is a potent GPR119-dependent GLP-1 secreting agent. Here we present a structure-activity relationship (SAR) study of a synthetic library of oleoyl-LPI mimetics capable to induce the physiological release of GLP-1 from gastrointestinal enteroendocrine cells (EECs). The best lead compounds have shown potent and efficient release of GLP-1 in vitro from human and murine cells, and in vivo in diabetic db/db mice. We have also generated a molecular model of oleoyl-LPI, as well as its best performing analogues, interacting with the orthosteric site of GPR119, laying foundational evidence for their pharmacological activity.

Switching Lysophosphatidylserine G Protein-Coupled Receptor Agonists to Antagonists by Acylation of the Hydrophilic Serine Amine.

Three human G protein-coupled receptors (GPCRs)-GPR34/LPS1, P2Y10/LPS2, and GPR174/LPS3-are activated specifically by lysophosphatidylserine (LysoPS), an endogenous hydrolysis product of a cell membrane component, phosphatidylserine (PS). LysoPS consists of l-serine, glycerol, and fatty acid moieties connected by phosphodiester and ester linkages. We previously generated potent and selective GPCR agonists by modification of the three modules and the ester linkage. Here, we show that a novel modification of the hydrophilic serine moiety, that is, N-acylations of the serine amine, converted a GPR174 agonist to potent GPR174 antagonists. Structural exploration of the amide functionality provided access to a range of activities from agonist to partial agonist to antagonist. The present study would provide a new strategy for the development of lysophospholipid receptor antagonists.

Lysophosphatidylglucoside is a GPR55 -mediated chemotactic molecule for human monocytes and macrophages.

Neutrophils undergo spontaneous apoptosis within 24-48 h after leaving bone marrow. Apoptotic neutrophils are subsequently phagocytosed and cleared by macrophages, thereby maintaining neutrophil homeostasis. Previous studies have demonstrated involvement of lysophosphatidylglucoside (lysoPtdGlc), a degradation product of PtdGlc, in modality-specific repulsive guidance of spinal sensory axons, via its specific receptor GPR55. In the present study, using human monocytic cell line THP-1 as a model, we demonstrated that lysoPtdGlc induces monocyte/macrophage migration with typical bell-haped curve and a peak at concentration 10-9 M. Lysophosphatidylinositol (lysoPtdIns), a known GPR55 ligand, induced migration at higher concentration (10-7 M). LysoPtdGlc-treated cells had a polarized shape, whereas lysoPtdIns-treated cells had a spherical shape. In EZ-TAXIScan (chemotaxis) assay, lysoPtdGlc induced chemotactic migration activity of THP-1 cells, while lysoPtdIns induced random migration activity. GPR55 antagonist ML193 inhibited lysoPtdGlc-induced THP-1 cell migration, whereas lysoPtdIns-induced migration was inhibited by CB2-receptor inverse agonist. SiRNA experiments showed that GPR55 mediated lysoPtdGlc-induced migration, while lysoPtdIns-induced migration was mediated by CB2 receptor. Our findings, taken together, suggest that lysoPtdGlc functions as a chemotactic molecule for human monocytes/macrophages via GPR55 receptor, while lysoPtdIns induces random migration activity via CB2 receptor.

2-Arachidonyl-lysophosphatidylethanolamine Induces Anti-Inflammatory Effects on Macrophages and in Carrageenan-Induced Paw Edema.

2-Arachidonyl-lysophosphatidylethanolamine, shortly 2-ARA-LPE, is a polyunsaturated lysophosphatidylethanolamine. 2-ARA-LPE has a very long chain arachidonic acid, formed by an ester bond at the sn-2 position. It has been reported that 2-ARA-LPE has anti-inflammatory effects in a zymosan-induced peritonitis model. However, it's action mechanisms are poorly investigated. Recently, resolution of inflammation is considered to be an active process driven by M2 polarized macrophages. Therefore, we have investigated whether 2-ARA-LPE acts on macrophages for anti-inflammation, whether 2-ARA-LPE modulates macrophage phenotypes to reduce inflammation, and whether 2-ARA-LPE is anti-inflammatory in a carrageenan-induced paw edema model. In mouse peritoneal macrophages, 2-ARA-LPE was found to inhibit lipopolysaccharide (LPS)-induced M1 macrophage polarization, but not induce M2 polarization. 2-ARA-LPE inhibited the inductions of inducible nitric oxide synthase and cyclooxygenase-2 in mouse peritoneal macrophages at the mRNA and protein levels. Furthermore, products of the two genes, nitric oxide and prostaglandin E2, were also inhibited by 2-ARA-LPE. However, 1-oleoyl-LPE did not show any activity on the macrophage polarization and inflammatory responses. The anti-inflammatory activity of 2-ARA-LPE was also verified in vivo in a carrageenan-induced paw edema model. 2-ARA-LPE inhibits LPS-induced M1 polarization, which contributes to anti-inflammation and suppresses the carrageenan-induced paw edema in vivo.

Sphingosine 1-Phosphate Liposomes for Targeted Nitric Oxide Delivery to Mediate Anticancer Effects against Brain Glioma Tumors.

Specifically targeting glioblastoma multiforme (GBM) blood vessels and actively enhancing the permeability of the brain-blood-tumor barrier (BBTB) are two extremely difficult challenges currently hindering the development of effective therapies against GBM. Herein, a liposome drug delivery system (S1P/JS-K/Lipo) is described, which delivers the nitric oxide (NO) prodrug JS-K, O2 -(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl) piperazin-1-yl] diazen-1-ium-1,2-diolate, to GBM tumors using sphingosine-1-phosphate (S1P)-signaling molecules as active targeting lipid ligands. It is revealed that S1P/JS-K/Lipo actively penetrates the BBTB, aided by caveolin-1-mediated transcytosis, and it is demonstrated that the system specifically interacts with S1P receptors (S1PRs), which are highly expressed on GBM cells. Nondestructive ultrasound imaging in GBM mouse models is also utilized to observe microsized NO bubble production from JS-K, as catalyzed by the glutathione S-transferases (GSTs) resident in GBM cells. Given that these NO bubbles strongly promote GBM cell death in vivo, the S1PR-targeted liposome delivery system-which successfully achieves BBTB penetration and tumor targeted delivery of a complex multicomponent drug regimen-represents a promising approach for targeted therapies against GBM and other carcinomas characterized by elevated S1PR expression.

Lysophosphatidic acid (LPA) receptor modulators: Structural features and recent development.

Lysophosphatidic acid (LPA) activates six LPA receptors (LPAR1-6) and regulates various cellular activities such as cell proliferation, cytoprotection, and wound healing. Many studies elucidated the pathological outcomes of LPA are due to the alteration in signaling pathways, which include migration and invasion of cancer cells, fibrosis, atherosclerosis, and inflammation. Current pathophysiological research on LPA and its receptors provides a means that LPA receptors are new therapeutic targets for disorders associated with LPA. Various chemical modulators are developed and are under investigation to treat a wide range of pathological complications. This review summarizes the physiological and pathological roles of LPA signaling, development of various LPA modulators, their structural features, patents, and their clinical outcomes.

Ceramides and Sphingosino-1-Phosphate in Obesity.

Obesity is a growing worldwide problem, especially in developed countries. This disease adversely affects the quality of life and notably contributes to the development of type 2 diabetes, metabolic syndrome, and cardiovascular disorders. It is characterised by excessive lipids accumulation in the subcutaneous and visceral adipose tissue. Considering the secretory function of adipose tissue, this leads to impaired adipokines and cytokines release. Changes in adipose tissue metabolism result in chronic inflammation, pancreatic islets dysfunction and peripheral insulin resistance. In addition to saturating various adipocytes, excess lipids are deposited into non-adipose peripheral tissues, which disturbs cell metabolism and causes a harmful effect known as lipotoxicity. Fatty acids are metabolised into bioactive lipids such as ceramides, from which sphingolipids are formed. Ceramides and sphingosine-1-phosphate (S1P) are involved in intracellular signalling, cell proliferation, migration, and apoptosis. Studies demonstrate that bioactive lipids have a crucial role in regulating insulin signalling pathways, glucose homeostasis and ß cell death. Data suggests that ceramides may have an opposite cellular effect than S1P; however, the role of S1P remains controversial. This review summarises the available data on ceramide and sphingolipid metabolism and their role in obesity.