Structural basis for lysophosphatidylserine recognition by GPR34.

GPR34 is a recently identified G-protein coupled receptor, which has an immunomodulatory role and recognizes lysophosphatidylserine (LysoPS) as a putative ligand. Here, we report cryo-electron microscopy structures of human GPR34-Gi complex bound with one of two ligands bound: either the LysoPS analogue S3E-LysoPS, or M1, a derivative of S3E-LysoPS in which oleic acid is substituted with a metabolically stable aromatic fatty acid surrogate. The ligand-binding pocket is laterally open toward the membrane, allowing lateral entry of lipidic agonists into the cavity. The amine and carboxylate groups of the serine moiety are recognized by the charged residue cluster. The acyl chain of S3E-LysoPS is bent and fits into the L-shaped hydrophobic pocket in TM4-5 gap, and the aromatic fatty acid surrogate of M1 fits more appropriately. Molecular dynamics simulations further account for the LysoPS-regioselectivity of GPR34. Thus, using a series of structural and physiological experiments, we provide evidence that chemically unstable 2-acyl LysoPS is the physiological ligand for GPR34. Overall, we anticipate the present structures will pave the way for development of novel anticancer drugs that specifically target GPR34.

Structural basis for ligand recognition and signaling of the lysophosphatidylserine receptors GPR34 and GPR174.

Lysophosphatidylserine (LysoPS) is a naturally occurring lipid mediator involved in various physiological and pathological processes especially those related to the immune system. GPR34, GPR174, and P2Y10 have been identified as the receptors for LysoPS, and its analogues have been developed as agonists or antagonists for these receptors. However, the lack of structural information hinders the drug development with novel characteristics, such as nonlipid ligands and allosteric modulators. Here, we determined the structures of human GPR34 and GPR174 in complex with LysoPS and G protein by cryo-EM. Combined with structural analysis and functional studies, we elucidated the lipid-binding modes of these receptors. By structural comparison, we identified the structural features of GPR34 and GPR174 in active state. Taken together, our findings provide insights into ligand recognition and signaling of LysoPS receptors and will facilitate the development of novel therapeutics for related inflammatory diseases and autoimmune diseases.

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.

Exploration of LPS2 agonist binding modes using the combination of a new hydrophobic scaffold and homology modeling.

Lysophosphatidylserine (LysoPS) is an endogenous pan-agonist of three G-protein coupled receptors (GPCRs): LPS1/GPR34, LPS2/P2Y10, and LPS3/GPR174, and we previously reported a series of LysoPS-based agonists of these receptors. Interestingly, we found that LPS1 agonist activity was very sensitive to structural change at the hydrophobic fatty acid moiety, whereas LPS2 agonist activity was not. Here, to probe the molecular basis of LPS2 agonist binding, we developed a new class of hydrophobic fatty acid surrogates having a biphenyl-ether scaffold. The LPS2 agonist activity of these compounds proved sensitive to molecular modification of the hydrophobic skeleton. Thus, we next constructed an LPS2 model by homology modeling and docking/molecular dynamics (MD) simulation, and validated it by means of SAR studies together with point mutations of selected receptor amino-acid residues. The putative ligand-binding site of LPS2 is Γ-shaped, with a hydrophilic site horizontally embedded in the receptor transmembrane helix bundles and a perpendicular hydrophobic groove adjoining transmembrane domains 4 and 5 that is open to the membrane bilayer. The binding poses of LPS2 agonists to this site are consistent with easy incorporation of various kinds of fatty acid surrogates. Structural development based on this model afforded a series of potent and selective LPS2 full agonists, which showed enhanced in vitro actin stress fiber formation effect.

Non-naturally Occurring Regio Isomer of Lysophosphatidylserine Exhibits Potent Agonistic Activity toward G Protein-Coupled Receptors.

Lysophosphatidylserine (LysoPS), an endogenous ligand of G protein-coupled receptors, consists of l-serine, glycerol, and fatty acid moieties connected by phosphodiester and ester linkages, respectively. An ester linkage of phosphatidylserine can be hydrolyzed at the 1-position or at the 2-position to give 2-acyl lysophospholipid or 1-acyl lysophospholipid, respectively. 2-Acyl lysophospholipid is in nonenzymatic equilibrium with 1-acyl lysophospholipid in vivo. On the other hand, 3-acyl lysophospholipid is not found, at least in mammals, raising the question of whether the reason for this might be that the 3-acyl isomer lacks the biological activities of the other isomers. Here, to test this idea, we designed and synthesized a series of new 3-acyl lysophospholipids. Structure-activity relationship studies of more than 100 "glycol surrogate" derivatives led to the identification of potent and selective agonists for LysoPS receptors GPR34 and P2Y10. Thus, the non-natural 3-acyl compounds are indeed active and appear to be biologically orthogonal with respect to the physiologically relevant 1- and 2-acyl lysophospholipids.

Probing the Hydrophobic Binding Pocket of G-Protein-Coupled Lysophosphatidylserine Receptor GPR34/LPS1 by Docking-Aided Structure-Activity Analysis.

The ligands of certain G-protein-coupled receptors (GPCRs) have been identified as endogenous lipids, such as lysophosphatidylserine (LysoPS). Here, we analyzed the molecular basis of the structure-activity relationship of ligands of GPR34, one of the LysoPS receptor subtypes, focusing on recognition of the long-chain fatty acid moiety by the hydrophobic pocket. By introducing benzene ring(s) into the fatty acid moiety of 2-deoxy-LysoPS, we explored the binding site's preference for the hydrophobic shape. A tribenzene-containing fatty acid surrogate with modifications of the terminal aromatic moiety showed potent agonistic activity toward GPR34. Computational docking of these derivatives with a homology modeling/molecular dynamics-based virtual binding site of GPR34 indicated that a kink in the benzene-based lipid surrogates matches the L-shaped hydrophobic pocket of GPR34. A tetrabenzene-based lipid analogue bearing a bulky tert-butyl group at the 4-position of the terminal benzene ring exhibited potent GPR34 agonistic activity, validating the present hydrophobic binding pocket model.

Structure-activity relationships of lysophosphatidylserine analogs as agonists of G-protein-coupled receptors GPR34, P2Y10, and GPR174.

Lysophosphatidylserine (LysoPS) is an endogenous lipid mediator generated by hydrolysis of membrane phospholipid phosphatidylserine. Recent ligand screening of orphan G-protein-coupled receptors (GPCRs) identified two LysoPS-specific human GPCRs, namely, P2Y10 (LPS2) and GPR174 (LPS3), which, together with previously reported GPR34 (LPS1), comprise a LysoPS receptor family. Herein, we examined the structure-activity relationships of a series of synthetic LysoPS analogues toward these recently deorphanized LysoPS receptors, based on the idea that LysoPS can be regarded as consisting of distinct modules (fatty acid, glycerol, and l-serine) connected by phosphodiester and ester linkages. Starting from the endogenous ligand (1-oleoyl-LysoPS, 1), we optimized the structure of each module and the ester linkage. Accordingly, we identified some structural requirements of each module for potency and for receptor subtype selectivity. Further assembly of individually structure-optimized modules yielded a series of potent and LysoPS receptor subtype-selective agonists, particularly for P2Y10 and GPR174.

Lysophosphatidylserine analogues differentially activate three LysoPS receptors.

Lysophosphatidylserine (1-oleoyl-2 R-lysophosphatidylserine, LysoPS) has been shown to have lipid mediator-like actions such as stimulation of mast cell degranulation and suppression of T lymphocyte proliferation, although the mechanisms of LysoPS actions have been elusive. Recently, three G protein-coupled receptors (LPS1/GPR34, LPS2/P2Y10 and LPS3/GPR174) were found to react specifically with LysoPS, raising the possibility that LysoPS serves as a lipid mediator that exerts its role through these receptors. Previously, we chemically synthesized a number of LysoPS analogues and evaluated them as agonists for mast-cell degranulation. Here, we used a transforming growth factor-α (TGFα) shedding assay to see if these LysoPS analogues activated the three LysoPS receptors. Modification of the serine moiety significantly reduced the ability of the analogues to activate the three LysoPS receptors, whereas modification of other parts resulted in loss of activity in receptor-specific manner. We found that introduction of methyl group to serine moiety (1-oleoyl-lysophosphatidylallothreonine) and removal of sn-2 hydroxyl group (1-oleoyl-2-deoxy-LysoPS) resulted in reduction of reactivity with LPS1 and LPS3, respectively. Accordingly, we synthesized a LysoPS analogue with the two modifications (1-oleoyl-2-deoxy-lysophosphatidylallothreonine) and found it to be an LPS2-selective agonist. These pharmacological tools will definitely help to identify the biological roles of these LysoPS receptors.

Towards selective lysophospholipid GPCR modulators.

G-protein-coupled receptors (GPCRs) that recognize the lysophospholipids (LPLs) are grouped into two phylogenetically distinct families: the endothelial differentiation gene (Edg) and non-Edg GPCRs. Owing to their more recent identification, and hindered by a lack of selective pharmacological tools, our understanding of the functions and signaling pathways of the non-Edg GPCRs is still in its infancy. Targeting the non-conserved allosteric binding sites of the LPL GPCRs shows particular promise for the development of selective modulators by structure-based drug design. However, only one Edg GPCR (S1PR1) structure has been determined to date, and it has low sequence identity with the non-Edg GPCRs (<20%). Thus, a representative structure of a non-Edg GPCR remains a pressing objective for selective structure-based drug design. Obtaining selective modulators targeting the non-Edg receptors would help to unravel the biology behind these novel GPCRs and potentially will support therapeutic treatment of diseases such as cancer, inflammation, and neuropsychiatric disorders.

Modulation of chemokines and allergic airway inflammation by selective local sphingosine-1-phosphate receptor 1 agonism in lungs.

Sphingosine-1-phosphate and its receptors have emerged as important modulators of the immune response. The sphingosine-1-phosphate prodrug 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) can alleviate experimental allergic airway inflammation. Nevertheless, the role of individual sphingosine-1-phosphate receptors in the regulation of allergic airway inflammation remains undefined. Using a newly characterized potent and selective sphingosine-1-phosphate receptor 1 (S1P1) agonist with physical properties allowing airway delivery, we studied the contribution of S1P1 signaling to eosinophilic airway inflammation induced in ovalbumin-immunized mice by airway challenges with ovalbumin. Airway delivery of receptor-nonselective sphingosine-1-phosphate prodrug significantly inhibits the sequential accumulation of antigen-presenting dendritic cells and CD4+ T cells in draining lymph nodes. This in turn suppressed by >80% the accumulation of CD4+ T cells and eosinophils in the airways. Systemic delivery of sphingosine-1-phosphate prodrug or of an S1P)1-specific agonist at doses sufficient to induce lymphopenia did not inhibit eosinophil accumulation in the airways. In contrast, local airway delivery of S1P1-specific agonist inhibited airways release of endogenous CCL5 and CCL17 chemokines, and significantly suppressed accumulation of activated T cells and eosinophils in the lungs. Specific S1P1 agonism in lungs contributes significantly to anti-inflammatory activities of sphingosine-1-phosphate therapeutics by suppressing chemokine release in the airways, and may be of clinical relevance.

International Union of Basic and Clinical Pharmacology. LXXVIII. Lysophospholipid receptor nomenclature.

Lysophospholipids are cell membrane-derived lipids that include both glycerophospholipids such as lysophosphatidic acid (LPA) and sphingoid lipids such as sphingosine 1-phosphate (S1P). These and related molecules can function in vertebrates as extracellular signals by binding and activating G protein-coupled receptors. There are currently five LPA receptors, along with a proposed sixth (LPA1-LPA6), and five S1P receptors (S1P1-S1P5). A remarkably diverse biology and pathophysiology has emerged since the last review, driven by cloned receptors and targeted gene deletion ("knockout") studies in mice, which implicate receptor-mediated lysophospholipid signaling in most organ systems and multiple disease processes. The entry of various lysophospholipid receptor modulatory compounds into humans through clinical trials is ongoing and may lead to new medicines that are based on this signaling system. This review incorporates IUPHAR Nomenclature Committee guidelines in updating the nomenclature for lysophospholipid receptors ( http://www.iuphar-db.org/DATABASE/FamilyMenuForward?familyId=36).

Pharmacological tools for lysophospholipid GPCRs: development of agonists and antagonists for LPA and S1P receptors.

Previous studies on lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) using various approaches have shown that both the molecules can act as intercellular signaling molecules. The discovery of the Edg subfamily of G-protein-coupled receptors (GPCRs) (later renamed LPA(1-3) and S1P(1-5)) for these molecules has opened up a new avenue for pathophysiological research on lysophospholipids. Genetic and molecular studies on lysophospholipid GPCRs have elucidated pathophysiological impacts and roles in cellular signaling pathways. Recently, lysophospholipid GPCR genes have been used to develop receptor subtype-selective agonists and antagonists. The discovery of FTY720, a novel immune modulator, along with other chemical tools, has provided a means of elucidating the functions of each lysophospholipid GPCR on an organ and the whole body level. This communication attempts to retrospectively review the development of agonists and antagonists for lysophospholipid GPCRs, provide integrated information on pharmacological tools for lysophospholipid GPCR signaling, and speculate on future drug development.

The enigmatic pharmacology of GPR55.

Preliminary data presented at conferences and in the patent literature introduced the possibility the orphan receptor GPR55 might account for some of the well-documented non-CB(1), non-CB(2) effects reported for certain cannabinoid ligands. Several peer-reviewed publications have recently emerged in which the pharmacology of the cannabinoids at GPR55 has been probed in more depth. Despite this, the classification of GPR55 as a cannabinoid receptor remains a contentious issue. The weight of evidence points to GPR55 as a receptor that is activated by certain cannabinoid ligands and by the bioactive lipid l-alpha-lysophosphatidylinsoitol. It couples to G(12) proteins, activates RhoA and mobilizes intracellular Ca(2+), possibly in an agonist- and tissue-dependant manner, thus displaying 'agonist functional selectivity'. Here, I review the recent literature in an effort to glean the key controversies and outstanding questions surrounding the interaction between cannabinoids and this orphan receptor.

Lysophospholipid receptors as potential drug targets in tissue transplantation and autoimmune diseases.

New therapies directed at ameliorating or altering autoimmune diseases represent an area of significant medical need. Included amongst autoimmune diseases are problems related to transplantation rejection, as well as a number of neurological diseases such as Multiple Sclerosis (MS). A new group of molecular targets that may lead to novel therapies are lysophospholipid (LP) receptors. A large range of biological activities has been attributed to the actions of these simple phospholipids that include well-studied members lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). Documented cellular effects of these lipid molecules encompass growth-factor-like influences on cells, including but not limited to survival, migration, adhesion differentiation, as well as pathophysiological actions associated with cancer. In turn, these cellular effects have roles in developing and adult organ systems such as the nervous system, cardiovascular system, reproductive system and, of relevance here, the immune system. The mechanisms for these actions can be attributed to a growing family of cognate, 7-transmembrane G protein-coupled receptors (GPCRs), with documented validation through studies utilizing pharmacology, molecular genetics and an enlarging repertoire of chemical tools having agonist or antagonist properties. The growing literature on immunological effects of LP receptors, particularly those mediating the effects of S1P, has suggested possible therapeutic roles for this class of receptors. In particular, entry into humans of a non-selective S1P receptor agonist, FTY720, for kidney transplantation and possibly other indications (e.g., Multiple Sclerosis), has raised prospects for efficacious treatment of human diseases based on LP receptor targets. Here we provide a brief introduction to receptor-mediated lysophospholipid signaling and discuss its basic and potential therapeutic roles in autoimmune-related diseases.