ST-2191, an Anellated Bismorpholino Derivative of Oxy-Fingolimod, Shows Selective S1P1 Agonist and Functional Antagonist Potency In Vitro and In Vivo.
TL;DR: A novel anellated bismorpholino derivative of oxy-fingolimod, named ST-2191, which exerts selective S1P1 agonist and functional antagonist potency and is effective in reducing the lymphocyte number in mice is reported on.
Abstract: Sphingosine 1-phosphate (S1P) is an extensively studied signaling molecule that contributes to cell proliferation, survival, migration and other functions through binding to specific S1P receptors. The cycle of S1P1 internalization upon S1P binding and recycling to the cell surface when local S1P concentrations are low drives T cell trafficking. S1P1 modulators, such as fingolimod, disrupt this recycling by inducing persistent S1P1 internalization and receptor degradation, which results in blocked egress of T cells from the secondary lymphoid tissues. The approval of these compounds for the treatment of multiple sclerosis has placed the development of S1PR modulators in the focus of pharmacological research, mostly for autoimmune indications. Here, we report on a novel anellated bismorpholino derivative of oxy-fingolimod, named ST-2191, which exerts selective S1P1 agonist and functional antagonist potency. ST-2191 is also effective in reducing the lymphocyte number in mice, and this effect is not dependent on phosphorylation by sphingosine kinase 2 for activity. These data show that ST-2191 is a novel S1P1 modulator, but further experiments are needed to analyze the therapeutic impact of ST-2191 in animal models of autoimmune diseases.
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TL;DR: The role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts is addressed, showing that sphingolipids have diverse effects onEpo synthesis.
Abstract: Erythropoietin (Epo) is a crucial hormone regulating red blood cell number and consequently the hematocrit. Epo is mainly produced in the kidney by interstitial fibroblast-like cells. Previously, we have shown that in cultures of the immortalized mouse renal fibroblast-like cell line FAIK F3-5, sphingosine 1-phosphate (S1P), by activating S1P1 and S1P3 receptors, can stabilize hypoxia-inducible factor (HIF)-2α and upregulate Epo mRNA and protein synthesis. In this study, we have addressed the role of intracellular iS1P derived from sphingosine kinases (Sphk) 1 and 2 on Epo synthesis in F3-5 cells and in mouse primary cultures of renal fibroblasts. We show that stable knockdown of Sphk2 in F3-5 cells increases HIF-2α protein and Epo mRNA and protein levels, while Sphk1 knockdown leads to a reduction of hypoxia-stimulated HIF-2α and Epo protein. A similar effect was obtained using primary cultures of renal fibroblasts isolated from wildtype mice, Sphk1−/−, or Sphk2−/− mice. Furthermore, selective Sphk2 inhibitors mimicked the effect of genetic Sphk2 depletion and also upregulated HIF-2α and Epo protein levels. The combined blockade of Sphk1 and Sphk2, using Sphk2−/− renal fibroblasts treated with the Sphk1 inhibitor PF543, resulted in reduced HIF-2α and Epo compared to the untreated Sphk2−/− cells. Exogenous sphingosine (Sph) enhanced HIF-2α and Epo, and this was abolished by the combined treatment with the selective S1P1 and S1P3 antagonists NIBR-0213 and TY52156, suggesting that Sph was taken up by cells and converted to iS1P and exported to then act in an autocrine manner through S1P1 and S1P3. The upregulation of HIF-2α and Epo synthesis by Sphk2 knockdown was confirmed in the human hepatoma cell line Hep3B, which is well-established to upregulate Epo production under hypoxia. In summary, these data show that sphingolipids have diverse effects on Epo synthesis. While accumulation of intracellular Sph reduces Epo synthesis, iS1P will be exported to act through S1P1+3 to enhance Epo synthesis. Furthermore, these data suggest that selective inhibition of Sphk2 is an attractive new option to enhance Epo synthesis and thereby to reduce anemia development in chronic kidney disease.
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TL;DR: In this article , a stable SPL-kd cell line of human podocytes was generated by the lentiviral shRNA transduction method and was characterized for reduced SPL mRNA and protein levels and increased S1P levels.
Abstract: Sphingosine 1-phosphate (S1P) lyase (SPL, Sgpl1) is an ER-associated enzyme that irreversibly degrades the bioactive lipid, S1P, and thereby regulates multiple cellular functions attributed to S1P. Biallelic mutations in the human Sglp1 gene lead to a severe form of a particular steroid-resistant nephrotic syndrome, suggesting that the SPL is critically involved in maintaining the glomerular ultrafiltration barrier, which is mainly built by glomerular podocytes. In this study, we have investigated the molecular effects of SPL knockdown (kd) in human podocytes to better understand the mechanism underlying nephrotic syndrome in patients. A stable SPL-kd cell line of human podocytes was generated by the lentiviral shRNA transduction method and was characterized for reduced SPL mRNA and protein levels and increased S1P levels. This cell line was further studied for changes in those podocyte-specific proteins that are known to regulate the ultrafiltration barrier. We show here that SPL-kd leads to the downregulation of the nephrin protein and mRNA expression, as well as the Wilms tumor suppressor gene 1 (WT1), which is a key transcription factor regulating nephrin expression. Mechanistically, SPL-kd resulted in increased total cellular protein kinase C (PKC) activity, while the stable downregulation of PKCδ revealed increased nephrin expression. Furthermore, the pro-inflammatory cytokine, interleukin 6 (IL-6), also reduced WT1 and nephrin expression. In addition, IL-6 caused increased PKCδ Thr505 phosphorylation, suggesting enzyme activation. Altogether, these data demonstrate that nephrin is a critical factor downregulated by the loss of SPL, which may directly cause podocyte foot process effacement as observed in mice and humans, leading to albuminuria, a hallmark of nephrotic syndrome. Furthermore, our in vitro data suggest that PKCδ could represent a new possible pharmacological target for the treatment of a nephrotic syndrome induced by SPL mutations.
TL;DR: S1P receptors are expressed throughout all mammalian organ systems as discussed by the authors , particularly S1P 1 , and their roles have been characterized through radioligand binding in heterologous networks.
Abstract: Abstract Sphingosine 1-phosphate (S1P) receptors ( nomenclature as agreed by the nomenclature as agreed by the NC-IUPHARNC-IUPHAR Subcommittee on Subcommittee on Lysophospholipid receptors [ Lysophospholipid receptors [9696]] ) are activated by the endogenous lipid sphingosine 1-phosphate (S1P). Originally cloned as orphan members of the endothelial differentiation gene ( edg ) family [16, 123], the receptors are currently designated as S1P 1 R through S1P 5 R [73, 16, 123]. Their gene nomenclature has been codified as human S1PR1, S1PR2, etc . (HUGO Gene Nomenclature Committee, HGNC) and S1pr1, S1pr2, etc . for mice (Mouse Genome Informatics Database, MGI) to reflect species and receptor function. All S1P receptors (S1PRs) have been knocked-out in mice constitutively and in some cases, conditionally. S1PRs, particularly S1P 1 , are expressed throughout all mammalian organ systems. Ligand delivery occurs via two known carriers (or "chaperones"): albumin and HDL-bound apolipoprotein M (ApoM), the latter of which elicits biased agonist signaling by S1P 1 in multiple cell types [18, 53]. The five S1PRs, two chaperones, and active cellular metabolism have complicated analyses of receptor ligand binding in native systems. Signaling pathways and physiological roles have been characterized through radioligand binding in heterologous
References
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TL;DR: It is shown that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolites of the immunosuppressive agent FTY720.
Abstract: Blood lymphocyte numbers, essential for the development of efficient immune responses, are maintained by recirculation through secondary lymphoid organs. We show that lymphocyte trafficking is altered by the lysophospholipid sphingosine-1-phosphate (S1P) and by a phosphoryl metabolite of the immunosuppressive agent FTY720. Both species were high-affinity agonists of at least four of the five S1P receptors. These agonists produce lymphopenia in blood and thoracic duct lymph by sequestration of lymphocytes in lymph nodes, but not spleen. S1P receptor agonists induced emptying of lymphoid sinuses by retention of lymphocytes on the abluminal side of sinus-lining endothelium and inhibition of egress into lymph. Inhibition of lymphocyte recirculation by activation of S1P receptors may result in therapeutically useful immunosuppression.
1,641 citations
"ST-2191, an Anellated Bismorpholino..." refers result in this paper
...In all S1PR-overexpressing cells, S1P dose-dependently activated the receptors, with effects already seen in the lower nM range, thus confirming previous studies of S1PR characterization [22,23]....
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TL;DR: The results suggest that FTY720, after phosphorylation, acts through sphingosine 1-phosphate signaling pathways to modulate chemotactic responses and lymphocyte trafficking.
1,482 citations
"ST-2191, an Anellated Bismorpholino..." refers result in this paper
...In all S1PR-overexpressing cells, S1P dose-dependently activated the receptors, with effects already seen in the lower nM range, thus confirming previous studies of S1PR characterization [22,23]....
[...]
TL;DR: Three-dimensional pharmacophore models are constructed, which are sufficiently selective to identify the described binding mode and are thus a useful tool for in-silico screening of large compound databases.
Abstract: From the historically grown archive of protein−ligand complexes in the Protein Data Bank small organic ligands are extracted and interpreted in terms of their chemical characteristics and features. Subsequently, pharmacophores representing ligand−receptor interaction are derived from each of these small molecules and its surrounding amino acids. Based on a defined set of only six types of chemical features and volume constraints, three-dimensional pharmacophore models are constructed, which are sufficiently selective to identify the described binding mode and are thus a useful tool for in-silico screening of large compound databases. The algorithms for ligand extraction and interpretation as well as the pharmacophore creation technique from the automatically interpreted data are presented and applied to a rhinovirus capsid complex as application example.
1,480 citations
University of California, San Diego1, Vanderbilt University Medical Center2, Georgia Institute of Technology3, Anschutz Medical Campus4, Duke University5, University of Texas Southwestern Medical Center6, Ochanomizu University7, University of Tokyo8, University of Graz9, Utrecht University10, University of California, Irvine11
TL;DR: A comprehensive classification of lipids with a common platform that is compatible with informatics requirements has been developed to deal with the massive amounts of data that will be generated by the lipid community.
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TL;DR: The International Lipid Classification and Nomenclature Committee under the sponsorship of the LIPID MAPS Consortium developed and established a "Comprehensive Classification System for Lipids" based on well defined chemical and biochemical principles and using an ontology that is extensible, flexible, and scalable as mentioned in this paper.
1,219 citations