Morpholino Analogues of Fingolimod as Novel and Selective S1P1 Ligands with In Vivo Efficacy in a Mouse Model of Experimental Antigen-Induced Encephalomyelitis.

Multiple sclerosis (MS) is a chronic, inflammatory, autoimmune disease of the central nervous system (CNS) which is associated with lower life expectancy and disability. The experimental antigen-induced encephalomyelitis (EAE) in mice is a useful animal model of MS, which allows exploring the etiopathogenetic mechanisms and testing novel potential therapeutic drugs. A new therapeutic paradigm for the treatment of MS was introduced in 2010 through the sphingosine 1-phosphate (S1P) analogue fingolimod (FTY720, Gilenya®), which acts as a functional S1P1 antagonist on T lymphocytes to deplete these cells from the blood. In this study, we synthesized two novel structures, ST-1893 and ST-1894, which are derived from fingolimod and chemically feature a morpholine ring in the polar head group. These compounds showed a selective S1P1 activation profile and a sustained S1P1 internalization in cultures of S1P1-overexpressing Chinese hamster ovary (CHO)-K1 cells, consistent with a functional antagonism. In vivo, both compounds induced a profound lymphopenia in mice. Finally, these substances showed efficacy in the EAE model, where they reduced clinical symptoms of the disease, and, on the molecular level, they reduced the T-cell infiltration and several inflammatory mediators in the brain and spinal cord. In summary, these data suggest that S1P1-selective compounds may have an advantage over fingolimod and siponimod, not only in MS but also in other autoimmune diseases.

Downregulation of S1P Lyase Improves Barrier Function in Human Cerebral Microvascular Endothelial Cells Following an Inflammatory Challenge.

Sphingosine 1-phosphate (S1P) is a key bioactive lipid that regulates a myriad of physiological and pathophysiological processes, including endothelial barrier function, vascular tone, vascular inflammation, and angiogenesis. Various S1P receptor subtypes have been suggested to be involved in the regulation of these processes, whereas the contribution of intracellular S1P (iS1P) through intracellular targets is little explored. In this study, we used the human cerebral microvascular endothelial cell line HCMEC/D3 to stably downregulate the S1P lyase (SPL-kd) and evaluate the consequences on endothelial barrier function and on the molecular factors that regulate barrier tightness under normal and inflammatory conditions. The results show that in SPL-kd cells, transendothelial electrical resistance, as a measure of barrier integrity, was regulated in a dual manner. SPL-kd cells had a delayed barrier build up, a shorter interval of a stable barrier, and, thereafter, a continuous breakdown. Contrariwise, a protection was seen from the rapid proinflammatory cytokine-mediated barrier breakdown. On the molecular level, SPL-kd caused an increased basal protein expression of the adherens junction molecules PECAM-1, VE-cadherin, and ß-catenin, increased activity of the signaling kinases protein kinase C, AMP-dependent kinase, and p38-MAPK, but reduced protein expression of the transcription factor c-Jun. However, the only factors that were significantly reduced in TNFα/SPL-kd compared to TNFα/control cells, which could explain the observed protection, were VCAM-1, IL-6, MCP-1, and c-Jun. Furthermore, lipid profiling revealed that dihydro-S1P and S1P were strongly enhanced in TNFα-treated SPL-kd cells. In summary, our data suggest that SPL inhibition is a valid approach to dampenan inflammatory response and augmente barrier integrity during an inflammatory challenge.

Targeting the S1P receptor signaling pathways as a promising approach for treatment of autoimmune and inflammatory diseases.

The past two decades of intense research have revealed a key role of the sphingolipid molecule sphingosine 1-phosphate (S1P) in regulating multiple physiological and pathophysiological processes including cell proliferation and survival, cell migration, inflammatory mediator synthesis and tissue remodeling. S1P mainly acts through five high-affinity G protein-coupled S1P receptors, which are ubiquitously expressed and mediate a complex network of signaling in a cell type dependent manner. S1P receptors have become an attractive pharmacological target to interfere with S1P-mediated cellular responses, which contribute to various autoimmune and inflammatory diseases. Pioneering in this field was the synthesis of FTY720 (fingolimod, Gilenya®) from myriocin, one of the metabolites of the fungus Isaria sinclairii known from traditional Chinese medicine for its antibacterial and energy boosting effect. Fingolimod turned out as a very potent immunomodulatory agent that subsequently passed all clinical trials successfully and is now approved for the treatment of relapsing-remitting multiple sclerosis. Pharmacologically, fingolimod was characterized as a non-selective agonist of all of the S1P receptors (S1PR), with the exception of S1P2, and in addition, as a selective S1P1 functional antagonist by induction of irreversible S1P1 internalization and degradation. Since proper lymphocyte trafficking depends on the expression of S1P1 on lymphocytes, the degradation of S1P1 leads to trapping and accumulation of lymphocytes in secondary lymphoid tissue, and consequently to a depletion of lymphocytes from the blood. Novel S1PR modulators are now being developed with a more selective receptor activation profile and improved pharmacokinetic characteristics. In this review, we will summarize the state-of-the-art approaches that target directly or indirectly S1P signaling and may be useful as novel strategies to treat autoimmune and inflammatory diseases.

Downregulation of the S1P Transporter Spinster Homology Protein 2 (Spns2) Exerts an Anti-Fibrotic and Anti-Inflammatory Effect in Human Renal Proximal Tubular Epithelial Cells.

Sphingosine kinase (SK) catalyses the formation of sphingosine 1-phosphate (S1P), which acts as a key regulator of inflammatory and fibrotic reactions, mainly via S1P receptor activation. Here, we show that in the human renal proximal tubular epithelial cell line HK2, the profibrotic mediator transforming growth factor ß (TGFß) induces SK-1 mRNA and protein expression, and in parallel, it also upregulates the expression of the fibrotic markers connective tissue growth factor (CTGF) and fibronectin. Stable downregulation of SK-1 by RNAi resulted in the increased expression of CTGF, suggesting a suppressive effect of SK-1-derived intracellular S1P in the fibrotic process, which is lost when SK-1 is downregulated. In a further approach, the S1P transporter Spns2, which is known to export S1P and thereby reduces intracellular S1P levels, was stably downregulated in HK2 cells by RNAi. This treatment decreased TGFß-induced CTGF and fibronectin expression, and it abolished the strong induction of the monocyte chemotactic protein 1 (MCP-1) by the pro-inflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1ß. Moreover, it enhanced the expression of aquaporin 1, which is an important water channel that is expressed in the proximal tubules, and reverted aquaporin 1 downregulation induced by IL-1ß/TNFα. On the other hand, overexpression of a Spns2-GFP construct increased S1P secretion and it resulted in enhanced TGFß-induced CTGF expression. In summary, our data demonstrate that in human renal proximal tubular epithelial cells, SK-1 downregulation accelerates an inflammatory and fibrotic reaction, whereas Spns2 downregulation has an opposite effect. We conclude that Spns2 represents a promising new target for the treatment of tubulointerstitial inflammation and fibrosis.