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Sphingosine-1-phosphate (S1P) receptor (S1PR) agonists, such as fingolimod (FTY720), alleviate nociception in preclinical pain models by either activation (agonism) or inhibition (functional antagonism) of S1PR type-1 (S1PR1). However, the dose-dependence and temporal relationship between reversal of nociception and modulation of S1PR1 signaling has not been systematically investigated. This study examined the relationship between FTY720-induced antinociception and S1PR1 adaptation using a sciatic nerve chronic constriction injury (CCI) model of neuropathic pain in male and female C57Bl/6J mice. Daily injections of FTY720 for 14 days dose-dependently reversed CCI-induced mechanical allodynia without tolerance development, and concomitantly resulted in a dose-dependent reduction of G-protein activation by the S1PR1-selective agonist SEW2871 in the lumbar spinal cord and brain. These findings indicate FTY720-induced desensitization of S1PR1 signaling coincides with its anti-allodynic effects. Consistent with this finding, a single injection of FTY720 reversed mechanical allodynia while concomitantly producing partial desensitization of S1PR1-stimulated G-protein activation in the CNS. However, mechanical allodynia returned 24-hr post injection, despite S1PR1 desensitization at that time, demonstrating a dissociation between these measures. Furthermore, CCI surgery led to elevations of sphingolipid metabolites, including S1P, which were unaffected by daily FTY720 administration, suggesting FTY720 reversed mechanical allodynia by targeting S1PR1 rather than sphingolipid metabolism. Supporting this hypothesis, acute administration of the S1PR1-selective agonist CYM-5442 mimicked the anti-allodynic effect of FTY720. In contrast, the S1PR1-selective antagonist NIBR-0213 prevented the anti-allodynic effect of FTY720, but NIBR-0213 given alone did not affect nociception. These results indicate that FTY720 alleviates CCI-induced allodynia through a mechanism distinct from functional antagonism., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Proximod is a selective agonist of sphingosine-1-phosphate receptor-1 (S1PR1). It acts by redirecting lymphocytes from the circulation to secondary lymph nodes, and is under development as an immunomodulator for rheumatoid arthritis. We aimed to evaluate the safety, pharmacokinetics, and preliminary efficacy of proximod in healthy volunteers and patients with rheumatoid arthritis., Methods: We did a two part, phase 1, double-blind, randomised, placebo-controlled, ascending dose trial at a single centre in China. Eligible participants were adults aged 18-50 years with a BMI of 18-28 kg/m 2 for healthy volunteers and aged 18-70 years with a BMI of 18-30 kg/m 2 for patients with rheumatoid arthritis. In part 1, healthy volunteers were randomly assigned within ten cohorts to receive a single oral dose of proximod (0·125 mg, 0·25 mg, 0·5 mg, 1 mg, 1·5 mg, 2 mg, 3 mg, 5 mg, 10 mg, or 15 mg in cohorts 1-10) or placebo. In part 2, healthy volunteers were randomly assigned to receive once-daily doses of proximod 5 mg or placebo, and patients with rheumatoid arthritis were randomly assigned to receive once-daily doses of proximod 5 mg, proximod 10 mg, or placebo, for 28 days. Patients and investigators were masked to treatment assignment. The primary outcomes were safety, tolerability, and pharmacokinetic profile of proximod for 72 days in healthy volunteers and for 48 days in patients with rhematoid arthritis, assessed in all treated participants. This trial is registered with ClinicalTrials.gov (NCT06361199, NCT06361186), and is complete., Findings: Between Nov 1, 2017, and June 22, 2021, 124 healthy volunteers were randomly assigned in part 1 of the study and 124 were included in the analyses (mean age 34·3 years [SD 6·9], 62 [50%] of 124 participants were women and 62 [50%] were men, and 116 [94%] were Han Chinese ethnicity). Between Feb 16, 2022, and Oct 8, 2023, 113 participants were screened for inclusion in part 2 (80 healthy volunteers and 33 patients with rheumatoid arthritis). 79 participants were excluded and 34 were randomly assigned (10 healthy participants and 24 patients with rheumatoid arthritis), 34 of whom were included in the analyses. Ten (100%) of ten healthy participants were Han Chinese ethnicity, with a mean age of 39·9 years (SD 7·3). Five (50%) of ten healthy volunteers were women and five (50%) were men). 22 (92%) of 24 participants with rheumatoid arthritis were Han Chinese ethnicity, with a mean age of 52·7 years (SD 6·8). 22 (92%) of 24 patients with rheumatoid arthritis were women and two (8%) were men. In part 1, all doses of proximod were well tolerated, with no dose-related adverse reactions or serious adverse events observed. In part 2, 74 adverse reactions were reported in eight (80%) of ten healthy volunteers and 22 (92%) of 24 patients with rheumatoid arthritis. Adverse events associated with proximod were predominantly mild or moderate. In part 2, the concentration of proximod and its active metabolite, proximod-phosphate, gradually increased in all three groups receiving proximod and the EC 50 of the S1PR1 agonist for proximod-phosphate (6·1 ng/mL) was reached on day 14 for both 5 mg groups, and on day 7 for the 10 mg group. The mean C trough values for proximod-phosphate on day 28 were 7·7 ng/mL and 10·2 ng/mL for 5 mg in healthy volunteers and patients with rheumatoid arthritis, respectively, and 15·3 ng/mL for 10 mg in patients with rheumatoid arthritis. In patients with rheumatoid arthritis, lymphocyte count decreased after treatment in all proximod groups reaching nadir at approximately day 28, with a corresponding percentage decline from baseline of 65·25% in the 5 mg group, 71·64% in the 10 mg group, and 20·57% in the placebo group., Interpretation: Proximod exhibited good tolerability over the 28-day treatment period, demonstrating its potential in reducing blood lymphocyte count. These results highlight the promise of the S1PR1 agonist proximod as a potential candidate for rheumatoid arthritis treatment, warranting further investigation in subsequent clinical studies., Funding: Beijing Union Pharmaceutical Factory and Jian Kuan (Suzhou) Biotechnology., Competing Interests: Declaration of interests YL, FY, YZ, XC, and JJ are employees of Beijing Union Pharmaceutical Factory, Peking Union Medical College and Jian Kuan (Suzhou) Biotechnology, which sponsored this study. The other authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved, including those for text and data mining, AI training, and similar technologies.)

Ocular abnormal angiogenesis and edema are featured in several ocular diseases. S1P signaling via S1P1 likely is part of the negative feedback mechanism necessary to maintain vascular health. In this study, we conducted pharmacological experiments to determine whether ASP4058, a sphingosine 1-phosphate receptor 1/5 (S1P1/5) agonist, is useful in abnormal vascular pathology in the eye. First, human retinal microvascular endothelial cells (HRMECs) were examined using vascular endothelial growth factor (VEGF)-induced cell proliferation and hyperpermeability. ASP4058 showed high affinity and inhibited VEGF-induced proliferation and hyperpermeability of HRMECs. Furthermore, S1P1 expression and localization changes were examined in the murine laser-induced choroidal neovascularization (CNV) model, a mouse model of exudative age-related macular degeneration, and the efficacy of ASP4058 was verified. In the CNV model mice, S1P1 tended to decrease in expression immediately after laser irradiation and colocalized with endothelial cells and Müller glial cells. Oral administration of ASP4058 also suppressed vascular hyperpermeability and CNV, and the effect was comparable to that of the intravitreal administration of aflibercept, an anti-VEGF drug. Next, efficacy was also examined in a retinal vein occlusion (RVO) model in which retinal vascular permeability was increased. ASP4058 dose-dependently suppressed the intraretinal edema. In addition, it suppressed the expansion of the perfusion area observed in the RVO model. ASP4058 also suppressed the production of VEGF in the eye. Collectively, ASP4058 can be a potential therapeutic agent that normalizes abnormal vascular pathology, such as age-related macular degeneration and RVO, through its direct action on endothelial cells., (© 2024. The Author(s).)

Primarily caused by chronic cigarette smoking (CS), emphysema is characterized by loss of alveolar cells comprising lung units involved in gas exchange and inflammation that culminate in airspace enlargement. Dysregulation of sphingolipid metabolism with increases of ceramide relative to sphingosine-1 phosphate (S1P) signaling has been shown to cause lung cell apoptosis and is emerging as a potential therapeutic target in emphysema. We sought to determine the impact of augmenting S1P signaling via S1P receptor 1 (S1P1) in a mouse model of CS-induced emphysema. DBA2 mice were exposed to CS for 4 or 6 mo and treated with pharmacological agonists of S1P1: phosphonated FTY720 (FTY720-1S and 2S analogs; 0.01-1.0 mg/kg) or GSK183303A (10 mg/kg). Pharmacological S1P1 agonists ameliorated CS-induced lung parenchymal apoptosis and airspace enlargement as well as loss of body weight. S1P1 agonists had modest inhibitory effects on CS-induced airspace inflammation and lung functional changes measured by Flexivent, improving lung tissue resistance. S1P1 abundance was reduced in chronic CS-conditions and remained decreased after CS-cessation or treatment with FTY720-1S. These results support an important role for S1P-S1P1 axis in maintaining the structural integrity of alveoli during chronic CS exposure and suggest that increasing both S1P1 signaling and abundance may be beneficial to counteract the effects of chronic CS exposure.

As a critical sphingolipid metabolite, sphingosine-1-phosphate (S1P) plays an essential role in immune and vascular systems. There are five S1P receptors, designated as S1PR1 to S1PR5, encoded in the human genome, and their activities are governed by endogenous S1P, lipid-like S1P mimics, or nonlipid-like therapeutic molecules. Among S1PRs, S1PR1 stands out due to its nonredundant functions, such as the egress of T and B cells from the thymus and secondary lymphoid tissues, making it a potential therapeutic target. However, the structural basis of S1PR1 activation and regulation by various agonists remains unclear. Here, we report four atomic resolution cryo-electron microscopy (cryo-EM) structures of Gi-coupled human S1PR1 complexes: bound to endogenous agonist d18:1 S1P, benchmark lipid-like S1P mimic phosphorylated Fingolimod [(S)-FTY720-P], or nonlipid-like therapeutic molecule CBP-307 in two binding modes. Our results revealed the similarities and differences of activation of S1PR1 through distinct ligands binding to the amphiphilic orthosteric pocket. We also proposed a two-step “shallow to deep” transition process of CBP-307 for S1PR1 activation. Both binding modes of CBP-307 could activate S1PR1, but from shallow to deep transition may trigger the rotation of the N-terminal helix of Gαi and further stabilize the complex by increasing the Gαi interaction with the cell membrane. We combine with extensive biochemical analysis and molecular dynamic simulations to suggest key steps of S1P binding and receptor activation. The above results decipher the common feature of the S1PR1 agonist recognition and activation mechanism and will firmly promote the development of therapeutics targeting S1PRs.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that mediates a wide spectrum of biological processes including apoptosis, immune response and inflammation. Here, we sought to understand how S1P signaling affects neuronal excitability in the central amygdala (CeA), which is a brain region associated with fear learning, aversive memory, and the affective dimension of pain. Because the G-protein coupled S1P receptor 1 (S1PR 1 ) has been shown to be the primary mediator of S1P signaling, we utilized S1PR 1 agonist SEW2871 and S1PR 1 antagonist NIBR to determine a potential role of S1PR 1 in altering the cellular physiology of neurons in the lateral division of the CeA (CeL) that share the neuronal lineage marker somatostatin (Sst). CeL-Sst neurons play a critical role in expression of conditioned fear and pain modulation. Here we used transgenic breeding strategies to identify fluorescently labeled CeL-Sst neurons for electrophysiological recordings. Using principal component analysis, we identified two primary subtypes of Sst neurons within the CeL in both male and female mice. We denoted the two types regular-firing (type A) and late-firing (type B) CeL-Sst neurons. In response to SEW2871 application, Type A neurons exhibited increased input resistance, while type B neurons displayed a depolarized resting membrane potential and voltage threshold, increased current threshold, and decreased voltage height. NIBR application had no effect on CeL Sst neurons, indicating the absence of tonic S1P-induced S1PR 1 . Our findings reveal subtypes of Sst neurons within the CeL that are uniquely affected by S1PR 1 activation, which may have implications for how S1P alters supraspinal circuits., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Brain-derived neurotrophic factor (BDNF) cannot cross the blood-brain barrier (BBB) when administered peripherally, which hinders its therapeutic potential. We utilized an in vitro BBB model-a tri-culture of a human endothelial cell line, a pericyte cell line, and an astrocyte cell line-to study the effect of twenty candidate lipophilic compounds on stimulating BDNF secretion in pericytes and astrocytes. The prostaglandin E2 receptor 4 agonist and sphingosine-1-phosphate receptor 5 agonist facilitated secretion of BDNF in the astrocyte, but did not decrease the transendothelial electrical resistance. These compounds may be promising agents for neurodegenerative and neuroinflammatory diseases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Aim: Heart failure with preserved ejection fraction (HFpEF) is a major cause of death worldwide with no approved treatment. Left ventricular hypertrophy (LVH) and diastolic dysfunction represent the structural and functional components of HFpEF, respectively. Endothelial dysfunction is prevalent in HFpEF and predicts cardiovascular events. We investigated if SAR247799, a G-protein-biased sphingosine-1-phosphate receptor 1 (S1P1) agonist with endothelial-protective properties, could improve cardiac and renal functions in a rat model of metabolic syndrome LVH and diastolic function., Methods: 31- and 65-week-old obese ZSF1 (Ob-ZSF1) rats, representing adult and aged animals with LVH and diastolic dysfunction, were randomized to a chow diet containing 0.025% (w/w) of SAR247799, or control (CTRL) chow for 4 weeks. Age-matched lean ZSF1 (Le-ZSF1) rats were fed control chow. Echocardiography, telemetry, biochemical and histological analysis were performed to evaluate the effect of SAR247799., Results: Echocardiography revealed that Ob-ZSF1 rats, in contrast to Le-ZSF1 rats, developed progressive diastolic dysfunction and cardiac hypertrophy with age. SAR247799 blunted the progression of diastolic dysfunction in adult and aged animals: in adult animals E/e' was evaluated at 21.8 ± 1.4 for Ob-ZSF1-CTRL, 19.5 ± 1.2 for Ob-ZSF1-SAR247799 p<0.01, and 19.5 ± 2.3 for Le-ZSF1-CTRL (median ± IQR). In aged animals E/e' was evaluated at 23.15 ± 4.45 for Ob-ZSF1-CTRL, 19.5 ± 5 for Ob-ZSF1-SAR247799 p<0.01, and 16.69 ± 1.7 for Le-ZSF1-CTRL, p<0.01 (median ± IQR). In aged animals, SAR247799 reduced cardiac hypertrophy (g/mm mean ± SEM of heart weight/tibia length 0.053 ± 0.001 for Ob-ZSF1-CTRL vs 0.046 ± 0.002 for Ob-ZSF1-SAR247799 p<0.01, Le-ZSF1-CTRL 0.035 ± 0.001) and myocardial perivascular collagen content (p<0.001), independently of any changes in microvascular density. In adult animals, SAR247799 improved endothelial function as assessed by the very low frequency bands of systolic blood pressure variability (mean ± SEM 67.8 ± 3.41 for Ob-ZSF1-CTRL 55.8 ± 4.27 or Ob-ZSF1-SAR247799, p<0.05 and 57.3 ± 1.82 Le-ZSF1-CTRL), independently of any modification of arterial blood pressure. In aged animals, SAR247799 reduced urinary protein/creatinine ratio, an index of glomerular injury, (10.3 ± 0.621 vs 8.17 ± 0.231 for Ob-ZSF1-CTRL vs Ob-ZSF1-SAR247799, respectively, p<0.05 and 0.294 ± 0.029 for Le-ZSF1-CTRL, mean ± SEM) and the fractional excretion of electrolytes. Circulating lymphocytes were not decreased by SAR247799, confirming lack of S1P1 desensitization., Conclusions: These experimental findings suggest that S1P1 activation with SAR247799 may be considered as a new therapeutic approach for LVH and diastolic dysfunction, major components of HFpEF., Competing Interests: Authors were employees of Sanofi at the time of conduct of the study and may have equity interest in Sanofi. BP, PJ and AAP are inventors of US patent number 9,782,411. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Sphingosine-1-phosphate (S1P) is an important bioactive lipid molecule in cell membrane metabolism and binds to G protein-coupled S1P receptors (S1PRs) to regulate embryonic development, physiological homeostasis, and pathogenic processes in various organs. S1PRs are lipid-sensing receptors and are therapeutic targets for drug development, including potential treatment of COVID-19. Herein, we present five cryo-electron microscopy structures of S1PRs bound to diverse drug agonists and the heterotrimeric Gi protein. Our structural and functional assays demonstrate the different binding modes of chemically distinct agonists of S1PRs, reveal the mechanical switch that activates these receptors, and provide a framework for understanding ligand selectivity and G protein coupling., (© 2021. The Author(s), under exclusive licence to Center for Excellence in Molecular Cell Science, CAS.)

Although the human immune response to cancer is naturally potent, it can be severely disrupted as a result of an immunosuppressive tumor microenvironment. Infiltrating regulatory T lymphocytes contribute to this immunosuppression by inhibiting proliferation of cytotoxic CD8 + T lymphocytes, which are key to an effective anti-cancer immune response. Other important contributory factors are thought to include metabolic stress caused by the local nutrient deprivation common to many solid tumors. Interleukin-33 (IL-33), an alarmin released in reaction to cell damage, and sphingosine-1-phosphate (S1P) are known to control cell positioning and differentiation of T lymphocytes. In an in vitro model of nutrient deprivation, we investigated the influence of IL-33 and S1P receptor 4 (S1P 4 ) on the differentiation and migration of human CD8 + T lymphocytes. Serum starvation of CD8 + T lymphocytes induced a subset of CD8 Low and IL-33 receptor-positive (ST2L + ) cells characterized by enhanced expression of the regulatory T cell markers CD38 and CD39. Both S1P 1 and S1P 4 were transcriptionally regulated after stimulation with IL-33. Moreover, expression of the chemokine receptor CXCR4 was increased in CD8 + T lymphocytes treated with the selective S1P 4 receptor agonist CYM50308. We conclude that nutrient deprivation promotes CD8 Low T lymphocytes, contributing to an immunosuppressive microenvironment and a poor anti-cancer immune response by limiting cytotoxic effector functions. Our results suggest that S1P 4 signaling modulation may be a promising target for anti-CXCR4 cancer immunotherapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Burkard, Dreis, Herrero San Juan, Huhn, Weigert, Pfeilschifter and Radeke.)

Metabolic activities are altered in cancer cells compared with those in normal cells, and the cancer-specific pathway becomes a potential therapeutic target. Higher cellular glucose consumption, which leads to lower glucose levels, is a hallmark of cancer cells. In an objective screening for chemicals that induce cell death under low-glucose conditions, we discovered a compound, denoted as ALESIA (Anticancer Ligand Enhancing Starvation-induced Apoptosis). By our shedding assay of transforming growth factor α in HEK293A cells, ALESIA was determined to act as a sphingosine-1-phosphate receptor 3-G 12 -biased agonist that promotes nitric oxide production and oxidative stress. The oxidative stress triggered by ALESIA resulted in the exhaustion of glucose, cellular NADPH deficiency, and then cancer cell death. Intraperitoneal administration of ALESIA improved the survival of mice with peritoneally disseminated rhabdomyosarcoma, indicating its potential as a new type of anticancer drug for glucose starvation therapy., Competing Interests: Declaration of interests M.T., I.K., S.Y., T.H., and M.H. filed patent PCT/JP2016/063381 for ALESIA. M.T., S.Y., T.H., and M.H. filed patent PCT/JP2017/039426 for ALESIA. H.O. is a chief operating officer of KinoPharma, Inc. M.H. is a founder, shareholder, and member of the scientific advisory board of KinoPharma, Inc., and BTB Drug Development Research Center Co., Ltd, although both companies are not directly involved in ALESIA. The other authors declare that they have no competing interests., (Copyright © 2021. Published by Elsevier Ltd.)

Ponesimod (PONVORY™) is an orally administered selective sphingosine-1-phosphate (S1P) receptor 1 (S1P 1 ) agonist being developed by the Janssen Pharmaceutical Companies of Johnson & Johnson for the treatment of multiple sclerosis (MS). Based on the results of the phase III OPTIMUM trial, ponesimod was recently approved in the USA for the treatment of relapsing forms of MS and has received a positive CHMP opinion in the EU for this indication. This article summarizes the milestones in the development of ponesimod leading to this first US approval.

Preeclampsia (PE), a hypertensive multisystem disorder, can lead to increased maternal and fetal mortality and morbidity. Sphingosine‑1‑phosphate (S1P) plays various roles, depending on the cell type, by binding to S1P receptors (S1PR). The present study evaluated the changes of S1PRs and investigated the potential role of S1PRs in pregnancy‑induced hypertension. PE rats were established by reduced uterine perfusion pressure. The involvement of S1PR2 was evaluated using JTE‑013, a specific S1PR2 antagonist, in PE rats. After the treatment, inflammatory cytokines were evaluated using enzyme linked immunosorbent assay, and the expression of vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS) activation and endothelial nitric oxide synthase (eNOS) were evaluated by reverse transcription‑quantitative PCR and western blotting. Results showed that S1PR2, but not S1PR1 and S1PR3, was significantly increased in the serum and placenta tissues of PE rats. Notably, JTE‑013 significantly decreased blood pressure, attenuated infiltration of inflammatory cells and decreased inflammation, as indicated by the decreased expression of inflammatory cytokines, including tumor necrosis factor‑α, interleukin‑1β (IL‑1β) and IL‑6, in placental tissues. Mechanistic studies demonstrated that JTE‑013 significantly increased the expression of VEGF and decreased the expression of fms‑like tyrosine kinase 1 in placental tissue. Furthermore, JTE‑013 prevented iNOS activation and increased eNOS in placental tissue. In summary, the present study demonstrated that S1PR2 contributed to hypertension and angiogenesis imbalance in PE rats.

Targeting the metastatic process to prevent disease dissemination in cancer remains challenging. One step in the metastatic cascade involves cancer cells transiting through the vascular endothelium after inflammation has increased the permeability of this cellular layer. Reducing inflammation-mediated gaps in the vascular endothelium could potentially be used to retard metastasis. This study describes the development of a novel ASR396-containing nanoparticle designed to activate the Sphingosine-1-Phosphate Receptor 1 (S1PR1) in order to tighten the junctions between the endothelial cells lining the vascular endothelium thereby inhibiting metastasis. ASR396 was derived from the S1PR1 agonist SEW2871 through chemical modification enabling the new compound to be loaded into a nanoliposome. ASR396 retained S1PR1 binding activity and the nanoliposomal formulation (nanoASR396) made it systemically bioavailable upon intravenous injection. Studies conducted in microvessels demonstrated that nanoASR396 significantly attenuated inflammatory mediator-induced permeability increase through the S1PR1 activation. Similarly, nanoASR396 inhibited gap formation mediated by inflammatory agents on an endothelial cell monolayer by decreasing levels of phosphorylated myosin light chain protein thereby inhibiting cellular contractility. In animal models, nanoASR396 inhibited lung metastasis by up to 80%, indicating its potential for retarding melanoma metastasis. Thus, a novel bioavailable nanoparticle-based S1PR1 agonist has been developed to negate the effects of inflammatory mediators on the vascular endothelium in order to reduce the metastatic dissemination of cancer cells., (Copyright © 2021. Published by Elsevier B.V.)

Sphingosine 1 phosphate (S1P) is a bioactive sphingolipid that exerts several functions in physiological and pathological conditions. The modulation of one of its receptors, S1P1, plays an important role in the egress of lymphocytes from lymph nodes and is a useful target in multiple sclerosis (MS) treatment. A new drug, siponimod (BAF-312) has been recently approved for the treatment of secondary progressive MS and has affinity for two S1P receptors, S1P1 and S1P5. The two receptors are expressed by endothelial cells that, as components of the blood-brain barrier (BBB), prevent the access of solutes and lymphocytes into the central nervous system, function often compromised in MS. Using an in vitro BBB model exposed to inflammatory cytokines (TNFα and IFNγ, 5 UI and 10 UI respectively), we evaluated the effects of BAF-312 (100 nM) on expression and function of endothelial tight junctional proteins (Zo-1 and claudin-5), regulation of transendothelial electrical resistance (TEER) and permeability to FITC-conjugated dextran. Zo-1 expression, as well as TEER values, were promptly recovered (24 h) when both S1P1 and S1P5 were activated by BAF-312. In contrast, at this time point, activation of S1P5 with the selective agonist UC-42-WP04 (300 nM) or with BAF-312, under blockade of S1P1 with the selective antagonist NIBR-0213 (1 μM), resulted in recovery of expression and localization of claudin-5 and reduction of TNFα/INFγ-induced expression of metalloproteinase 9. Only after a prolonged BAF-312 exposure (48 h), S1P1 was involved through activation of the PI3K/Akt pathway. The PI3K inhibitor LY294002 (10 µM) prevented in fact the effects of BAF-312 on all the parameters examined. In conclusion, BAF-312, by modulating both S1P1 and S1P5, may strengthen BBB properties, thus providing additional effects in the treatment of MS., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The sphingosine 1-phosphate (S1P) receptor 1 (S1P 1 ) has emerged as a therapeutic target for the treatment of multiple sclerosis (MS). Fingolimod (FTY720) is the first functional antagonist of S1P 1 that has been approved for oral treatment of MS. Previously, we have developed novel butterfly derivatives of FTY720 that acted similar to FTY720 in reducing disease symptoms in a mouse model of experimental autoimmune encephalomyelitis (EAE). In this study, we have synthesized a piperidine derivative of the oxazolo-oxazole compounds, denoted ST-1505, and its ring-opened analogue ST-1478, and characterised their in-vitro and in-vivo functions. Notably, the 3-piperidinopropyloxy moiety resembles a structural motif of pitolisant, a drug with histamine H 3 R antagonistic/inverse agonist activity approved for the treatment of narcolepsy. Both novel compounds exerted H 3 R affinities, and in addition, ST-1505 was characterised as a dual S1P 1+3 agonist, whereas ST-1478 was a dual S1P 1+5 agonist. Both multitargeting compounds were also active in mice and reduced the lymphocyte numbers as well as diminished disease symptoms in the mouse model of MS. The effect of ST-1478 was dependent on SK-2 activity suggesting that it is a prodrug like FTY720, but with a more selective S1P receptor activation profile, whereas ST-1505 is a fully active drug even in the absence of SK-2. In summary, these data suggest that the well soluble piperidine derivatives ST-1505 and ST-1478 hold promise as novel drugs for the treatment of MS and other autoimmune or inflammatory diseases, and by their H 3 R antagonist potency, they might additionally improve cognitive impairment during disease., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator approved for treating multiple sclerosis, is reported to prevent excitotoxic insult. Because excessive glutamate release is a major cause of neuronal damage in various neurological disorders, the effect of fingolimod on glutamate release in rat cerebrocortical nerve terminals (synaptosomes) was investigated in the current study. Fingolimod decreased 4-aminopyridine (4-AP)-stimulated glutamate release and calcium concentration elevation. Fingolimod-mediated inhibition of 4-AP-induced glutamate release was dependent on extracellular calcium, persisted in the presence of the glutamate transporter inhibitor DL-TBOA or intracellular Ca 2+ -releasing inhibitors dantrolene and CGP37157, and was prevented by blocking vesicular transporters or N- and P/Q-type channels. Western blot and immunocytochemical analysis revealed the presence of S1P1 receptor proteins in presynaptic terminals. Fingolimod-mediated inhibition of 4-AP-induced glutamate release was also abolished by the sphingosine kinase inhibitor DMS, selective S1P1 receptor antagonist W146, Gi/o protein inhibitor pertussis toxin, and G protein βγ subunit inhibitor gallein; however, it was unaffected by the adenylyl cyclase inhibitor SQ22536, protein kinase A inhibitor H89, and phospholipase C inhibitor U73122. These data indicate that fingolimod decreases glutamate release from rat cerebrocortical synaptosomes by suppressing N- and P/Q-type Ca 2+ channel activity; additionally, the activation of presynaptic S1P1 receptors and the G protein βγ subunit participates in achieving the effect., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Rationale: Cerebrovascular function is critical for brain health, and endogenous vascular protective pathways may provide therapeutic targets for neurological disorders. S1P (Sphingosine 1-phosphate) signaling coordinates vascular functions in other organs, and S1P 1 (S1P receptor-1) modulators including fingolimod show promise for the treatment of ischemic and hemorrhagic stroke. However, S1P 1 also coordinates lymphocyte trafficking, and lymphocytes are currently viewed as the principal therapeutic target for S1P 1 modulation in stroke., Objective: To address roles and mechanisms of engagement of endothelial cell S1P 1 in the naive and ischemic brain and its potential as a target for cerebrovascular therapy., Methods and Results: Using spatial modulation of S1P provision and signaling, we demonstrate a critical vascular protective role for endothelial S1P 1 in the mouse brain. With an S1P 1 signaling reporter, we reveal that abluminal polarization shields S1P 1 from circulating endogenous and synthetic ligands after maturation of the blood-neural barrier, restricting homeostatic signaling to a subset of arteriolar endothelial cells. S1P 1 signaling sustains hallmark endothelial functions in the naive brain and expands during ischemia by engagement of cell-autonomous S1P provision. Disrupting this pathway by endothelial cell-selective deficiency in S1P production, export, or the S1P 1 receptor substantially exacerbates brain injury in permanent and transient models of ischemic stroke. By contrast, profound lymphopenia induced by loss of lymphocyte S1P 1 provides modest protection only in the context of reperfusion. In the ischemic brain, endothelial cell S1P 1 supports blood-brain barrier function, microvascular patency, and the rerouting of blood to hypoperfused brain tissue through collateral anastomoses. Boosting these functions by supplemental pharmacological engagement of the endothelial receptor pool with a blood-brain barrier penetrating S1P 1 -selective agonist can further reduce cortical infarct expansion in a therapeutically relevant time frame and independent of reperfusion., Conclusions: This study provides genetic evidence to support a pivotal role for the endothelium in maintaining perfusion and microvascular patency in the ischemic penumbra that is coordinated by S1P signaling and can be harnessed for neuroprotection with blood-brain barrier-penetrating S1P 1 agonists.

Lysophospholipids are a class of bioactive lipid molecules that produce their effects through various G protein-coupled receptors (GPCRs). Sphingosine 1-phosphate (S1P) is perhaps the most studied lysophospholipid and has a role in a wide range of physiological and pathophysiological events, via signalling through five distinct GPCR subtypes, S1PR 1 to S1PR 5 . Previous and continuing investigation of the S1P pathway has led to the approval of three S1PR modulators, fingolimod, siponimod and ozanimod, as medicines for patients with multiple sclerosis (MS), as well as the identification of new S1PR modulators currently in clinical development, including ponesimod and etrasimod. S1PR modulators have complex effects on S1PRs, in some cases acting both as traditional agonists as well as agonists that produce functional antagonism. S1PR subtype specificity influences their downstream effects, including aspects of their benefit:risk profile. Some S1PR modulators are prodrugs, which require metabolic modification such as phosphorylation via sphingosine kinases, resulting in different pharmacokinetics and bioavailability, contrasting with others that are direct modulators of the receptors. The complex interplay of these characteristics dictates the clinical profile of S1PR modulators. This review focuses on the S1P pathway, the characteristics and S1PR binding profiles of S1PR modulators, the mechanisms of action of S1PR modulators with regard to immune cell trafficking and neuroprotection in MS, together with a summary of the clinical effectiveness of the S1PR modulators that are approved or in late-stage development for patients with MS. Sphingosine 1-phosphate receptor modulator therapy for multiple sclerosis: differential downstream receptor signalling and clinical profile effects (MP4 65540 kb).

Many intracellular proteins are modified by N-acetylglucosamine, a post-translational modification termed O-GlcNAc. This modification is found on serine and threonine side chains and has the potential to regulate signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such example. We find that O-GlcNAc levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P), culminating in the phosphorylation of myosin light chain (MLC) and cellular contraction. Specifically, O-GlcNAc modification of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Finally, we demonstrate that O-GlcNAc levels alter the sensitivity of primary human dermal fibroblasts in a collagen-matrix model of wound healing. Our findings have important implications for the role of O-GlcNAc in fibroblast motility and differentiation, particularly in diabetic wound healing.

The discovery of novel antivirals to treat hepatitis B virus (HBV) infection is urgently needed, as the currently available drugs mainly target viral proteins at replication step, whereas host factors also play significant roles in HBV infection. Although numerous studies have reported candidate drugs for HBV treatment, there remains a need to find a new drug that may target other steps of the HBV life cycle. In this study, by drug screening of a 533 G-protein-coupled receptors (GPCRs)-associated compound library, we identified ponesimod, a selective agonist of sphingosine-1-phosphate receptor 1 (S1P 1 ), as a drug candidate for the suppression of HBV infection. However, the anti-HBV effect of ponesimod is independent of S1P 1 and other sphingosine-1-phosphate receptors (S1PRs). Treatment with ponesimod at an early step of infection but not at a post-entry step significantly reduced the HBV relaxed circular DNA (rcDNA) level in a dose-dependent manner. Ponesimod treatment did not inhibit attachment, binding, or internalization of HBV particles via endocytosis through an interaction with sodium taurocholate cotransporting polypeptide (NTCP) or epidermal growth factor receptor (EGFR). Importantly, during the transportation of HBV particles to the nucleus, co-localization of HBV with early endosomes but not with late endosomes and lysosomes was induced by the treatment with ponesimod, suggesting that ponesimod interferes with the conversion of early endosomes to late endosomes without significant damage to cellular growth. Conclusion: Ponesimod is a promising anti-HBV drug targeting the endosome maturation of HBV. This finding can be applied to the development of novel antivirals that target the trafficking pathway of HBV particles., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Neuroinflammation is a central part of cerebral ischemia/reperfusion injury. The novel immune suppressant, fingolimod, is a promising candidate to ameliorate stroke-induced damage. Fingolimod is efficacious in experimental ischemic models, but a rigorous meta-analysis is lacking that considers how different experiment variables affect outcomes. Methods: We conducted a systematic literature review of fingolimod in stroke models, with the aim of rigorously evaluating fingolimod's effects on reducing infarct volume improving neurological outcomes. Seventeen variables were evaluated as covariates for the source of heterogeneity, and effect sizes were combined by using normalized mean difference meta-analysis to evaluate efficacy. Study quality was evaluated by the CAMARADES ten-item checklist, and publication bias was evaluated by funnel plots and Egger's tests. Results: About 123 unduplicated articles were identified in the literature research. Of these papers, 118 articles were excluded after reading titles and abstracts. Another 17 articles were selected in this study. Study quality was moderate (median = 6; interquartile range = 4), and publication bias was statistically insignificant. fingolimod reduced infarct volume by 30.4% (95% CI 22.4%-38.3%; n  = 24; I 2 = 90.0%; p  < 0.0001) and consistently enhanced neurobehavioral outcome by 34.2% (95% CI 23.1%-45.2%; n  = 14; I 2 = 76.5%; p  < 0.0001). No single factors accounted for heterogeneity. Conclusions: Our rigorous statistical evaluation confirmed the neuroprotective properties of fingolimod. New data can be used in designing future clinical trials.

ONO-4641, 1-({6-[(2-methoxy-4-propylbenzyl)oxy]-1-methyl-3,4-dihydronaphthalen-2-yl}methyl)azetidine-3-carboxylic acid (ceralifimod), is a second-generation sphingosine 1-phosphate receptor agonist selective for sphingosine 1-phosphate receptors 1 and 5, and has clinical effects in multiple sclerosis. The objective of the present study was to explore other potential indications for ONO-4641 based on its immunomodulatory effects. ONO-4641 was tested in non-obese diabetic (NOD) mice, an animal model of spontaneous type 1 diabetes mellitus, an autoimmune disease with unmet medical needs. ONO-4641 at a dose of 0.1 mg/kg prevented the onset of diabetes mellitus in NOD mice. Furthermore, ONO-4641 at doses of 0.03 and 0.1 mg/kg decreased diabetic prevalence in NOD mice after the onset of diabetes mellitus in a dose-dependent manner. Histopathological analysis demonstrated that insulin-positive areas in the islets of mice administered 0.03 and 0.1 mg/kg ONO-4641 showed a tendency of high values although they were not significantly different from the Control group, which was treated with vehicle. These observations suggest ONO-4641 may delay the onset and progression of type 1 diabetes mellitus.

IMMH001, which is a prodrug for sphingosine-1-phosphate receptor 1 (S1P 1 ) agonist, is converted to the active form, its monophosphate ester (S)-IMMH001-P, by sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2) in vivo. In this study, we designed head-piece-modified analogues of IMMH001 based on structural information and prepared them with an efficient modular synthetic strategy. The analogues showed higher phosphorylation rates in human blood than the parent compound. These results indicated that the pro-R hydroxymethyl in the head-piece-moiety of IMMH001 prevents the pro-S hydroxymethyl from being phosphorylated by the kinase and ATP. The analogues may have better therapeutic potential., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Sphingosine-1 phosphate receptor-1 (S1P 1 ) activation maintains endothelial barrier integrity, whereas S1P 1 desensitization induces peripheral blood lymphopenia. The latter is exploited in the approval and/or late-stage development of receptor-desensitizing agents targeting the S1P 1 receptor in multiple sclerosis, such as siponimod, ozanimod, and ponesimod. SAR247799 is a recently described G protein-biased S1P 1 agonist that activates S1P 1 without desensitization and thus has endothelial-protective properties in patients without reducing lymphocytes. As SAR247799 demonstrated endothelial-protective effects at sub-lymphocyte-reducing doses, the possibility exists that other S1P 1 modulators could also exhibit endothelial-protective properties at lower doses. To explore this possibility, we sought to quantitatively compare the biased properties of SAR247799 with the most advanced clinical molecules targeting S1P 1 . In this study, we define the β-arrestin pathway component of the impedance profile following S1P 1 activation in a human umbilical vein endothelial cell line (HUVEC) and report quantitative indices of the S1P 1 activation-to-desensitization ratio of various clinical molecules. In a label-free impedance assay assessing endothelial barrier integrity and disruption, the mean estimates (95% confidence interval) of the activation-to-desensitization ratios of SAR247799, ponesimod, ozanimod, and siponimod were 114 (91.1-143), 7.66 (3.41-17.2), 6.35 (3.21-12.5), and 0.170 (0.0523-0.555), respectively. Thus, we show that SAR247799 is the most G protein-biased S1P 1 agonist currently characterized. This rank order of bias among the most clinically advanced S1P 1 modulators provides a new perspective on the relative potential of these clinical molecules for improving endothelial function in patients in relation to their lymphocyte-reducing (desensitization) properties., (© 2020 Sanofi R & D, Febs Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Dysregulated bile acid (BA) homeostasis is an extremely significant pathological phenomenon of intrahepatic cholestasis, and the accumulated BA could further trigger hepatocyte injury. Here, we showed that the expression of sphingosine-1-phosphate receptor 1 (S1PR1) was down-regulated by α-naphthylisothiocyanate (ANIT) in vivo and in vitro. The up-regulated S1PR1 induced by SEW2871 (a specific agonist of S1PR1) could improve ANIT-induced deficiency of hepatocyte tight junctions (TJs), cholestatic liver injury and the disrupted BA homeostasis in mice. BA metabolic profiles showed that SEW2871 not only reversed the disruption of plasma BA homeostasis, but also alleviated BA accumulation in the liver of ANIT-treated mice. Further quantitative analysis of 19 BAs showed that ANIT increased almost all BAs in mice plasma and liver, all of which were restored by SEW2871. Our data demonstrated that the top performing BAs were taurine conjugated bile acids (T-), especially taurocholic acid (TCA). Molecular mechanism studies indicated that BA transporters, synthetase, and BAs nuclear receptors (NRs) might be the important factors that maintained BA homeostasis by SEW2871 in ANIT-induced cholestasis. In conclusion, these results demonstrated that S1PR1 selective agonists might be the novel and potential effective agents for the treatment of intrahepatic cholestasis by recovering dysregulated BA homeostasis., Competing Interests: Declaration of Competing Interest The authors declared that no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The incidence and prevalence of ulcerative colitis are increasing globally. Although the exact cause and pathogenesis of this disease is unclear, research has led to a better understanding of the condition and to identification of new targets for therapy, which in turn has encouraged the development of new therapies. As well as biologic therapies, which have changed the way inflammatory bowel disease is managed, small molecules have been developed for the treatment of ulcerative colitis. These small molecule treatments are orally administered and are likely to bring a substantial shift in the way this chronic disease is treated. Oral therapies offer many advantages over infusion therapies, such as ease of use, increased acceptability by patients, and reduction of cost. This Review focuses not only on oral therapies that have been approved for use in ulcerative colitis, but also on those that are in development, providing a comprehensive overview for clinicians of available oral therapies and drugs that are likely to become available. We have also reviewed drugs that have shown promise in preclinical studies and could be effective future therapies., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Endothelial dysfunction is a hallmark of tissue injury and is believed to initiate the development of vascular diseases. Sphingosine-1 phosphate receptor-1 (S1P 1 ) plays fundamental physiological roles in endothelial function and lymphocyte homing. Currently available clinical molecules that target this receptor are desensitizing and are essentially S1P 1 functional antagonists that cause lymphopenia. They are clinically beneficial in autoimmune diseases such as multiple sclerosis. In patients, several side effects of S1P 1 desensitization have been attributed to endothelial damage, suggesting that drugs with the opposite effect, namely, the ability to activate S1P 1, could help to restore endothelial homeostasis. We found and characterized a biased agonist of S1P 1 , SAR247799, which preferentially activated downstream G protein signaling to a greater extent than β-arrestin and internalization signaling pathways. SAR247799 activated S1P 1 on endothelium without causing receptor desensitization and potently activated protection pathways in human endothelial cells. In a pig model of coronary endothelial damage, SAR247799 improved the microvascular hyperemic response without reducing lymphocyte numbers. Similarly, in a rat model of renal ischemia/reperfusion injury, SAR247799 preserved renal structure and function at doses that did not induce S1P 1 -desensitizing effects, such as lymphopenia and lung vascular leakage. In contrast, a clinically used S1P 1 functional antagonist, siponimod, conferred minimal renal protection and desensitized S1P 1 These findings demonstrate that sustained S1P 1 activation can occur pharmacologically without compromising the immune response, providing a new approach to treat diseases associated with endothelial dysfunction and vascular hyperpermeability., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

IMMH002 (1), a prodrug for a sphingosine-1-phosphate receptor 1 (S1P 1 ) agonist, is converted to the monophosphate ester, which has an immunomodulatory effect. Starting from prochiral amino alcohol 1, racemic and enantiomerically pure phosphates of 1 were synthesized. Pure enantiomers were obtained after the chiral resolution of the key intermediate by chiral high-performance liquid chromatography and the absolute configuration was determined by circular dichroism. In the in vitro homogeneous time-resolved fluorescence-IP1 functional assay, the (S)-enantiomer showed much higher S1P 1 activity and selectivity than the (R)-enantiomer. In the pharmacokinetic study, the ex vivo o-phthaldialdehyde derivatization protocol showed that the phosphate of 1 in rats was the S-configured enantiomer with >99% enantiomeric excess., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Future treatments of multiple sclerosis (MS), a chronic autoimmune neurodegenerative disease of the central nervous system (CNS), aim for simultaneous early targeting of peripheral immune function and neuroinflammation. Sphingosine-1-phosphate (S1P) receptor modulators are among the most promising drugs with both "immunological" and "non-immunological" actions. Selective S1P receptor modulators have been recently approved for MS and shown clinical efficacy in its mouse model, the experimental autoimmune encephalomyelitis (EAE). Here, we investigated the anti-inflammatory/neuroprotective effects of ozanimod (RPC1063), a S1P 1/5 modulator recently approved in the United States for the treatment of MS, by performing ex vivo studies in EAE brain. Electrophysiological experiments, supported by molecular and immunofluorescence analysis, revealed that ozanimod was able to dampen the EAE glutamatergic synaptic alterations, through attenuation of local inflammatory response driven by activated microglia and infiltrating T cells, the main CNS-cellular players of EAE synaptopathy. Electrophysiological studies with selective S1P 1 (AUY954) and S1P 5 (A971432) agonists suggested that S1P 1 modulation is the main driver of the anti-excitotoxic activity mediated by ozanimod. Accordingly, in vivo intra-cerebroventricular treatment of EAE mice with AUY954 ameliorated clinical disability. Altogether these results strengthened the relevance of S1P 1 agonists as immunomodulatory and neuroprotective drugs for MS therapy.

Introduction : Sphingosine-1-phosphate (S1P) is a membrane-derived lysophospholipid signaling molecule implicated in various physiological and pathological processes, such as regulation of the immune, cardiovascular, pulmonary, and nervous systems and theoretical cancer-related risks, through extracellular activation of S1P1-5 receptors. Areas covered : S1P receptor agonism is a novel strategy for the treatment of UC targeting lymphocyte recirculation, through blockade of lymphocyte egress from lymph nodes. We conducted an extensive literature review on PUBMED on currently available data on molecular aspects of S1P modulation, the mechanisms of action of S1PR agonists (fingolimod, ozanimod, etrasimod, and KRP-203), and their potential efficacy and safety for the treatment of patients with ulcerative colitis. Expert opinion : Selective S1P modulators have emerged to enlarge the efficacy and safety profile of this class of agents. Phase 3 programs should add the potential body of evidence to prove their benefit for the management of UC patients.

Sphingosine-1-phosphate (S1P) receptor 1 subtype (S1PR1) activation by its ligand S1P in the dorsal horn of the spinal cord causes mechanohypersensitivity. The cellular and molecular pathways remain poorly understood. We report that the activation of S1PR1 with an intrathecal injection of the highly selective S1PR1 agonist SEW2871 led to the development of mechanoallodynia by activating the nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome (increased expression of NLRP3, cleaved caspase 1 and mature IL-1β) in the dorsal horn of the spinal cord. The functional S1PR1 antagonist FTY720 blocked NLRP3 activation and IL-1β production. Moreover, inhibiting IL-10 signaling with an intrathecal injection of an anti-IL-10 antibody attenuated the beneficial effects exerted by FTY720. This finding suggests that disrupting S1PR1 signaling engages beneficial IL-10-dependent pathways. Notably, we found that mice with astrocyte-specific deletions of S1pr1 did not develop mechanoallodynia after intrathecal injection of SEW2871 and exhibited decreased levels of cleaved caspase 1, identifying astrocytes as a key cellular locus for S1PR1 activity. Our findings provide novel mechanistic insights on how S1PR1 activation in the spinal cord contributes to the development of nociception while identifying the cellular substrate for these activities. PERSPECTIVE: This study is the first to link the activation of NLRP3 and IL-1β signaling in the spinal cord and S1PR1 signaling in astrocytes to the development of S1PR1-evoked mechanoallodynia. These findings provide critical basic science insights to support the development of therapies targeted toward S1PR1., (Copyright © 2019 the American Pain Society. Published by Elsevier Inc. All rights reserved.)

Background and Purpose: Osteoporosis is a worldwide epidemic but pharmacological agents to stimulate new bone formation are scarce. We have shown that increasing tissue levels of sphingosine-1-phosphate (S1P) by blocking its degradation by the S1P lyase has pronounced osteoanabolic effect in mouse osteoporosis models by stimulating osteoblast differentiation through the S1P receptor 2 (S1P2). However, S1P lyase inhibitors have side effects complicating potential clinical use. Here, we tested whether direct S1P2 engagement by the S1P2 agonist CYM5520 exerted osteoanabolic potential in estrogen deficiency-induced osteopenia in mice. We compared its efficacy to LX2931, a novel S1P lyase inhibitor currently tested in rheumatoid arthritis., Experimental Approach: CYM5520, LX2931 or vehicle were administered to ovariectomized mice for 6 weeks beginning 5 weeks after ovariectomy, Bone mass, cellular composition and mechanical strength were assessed by microCT, histomorphometry and three point bending tests. Plasma markers of bone metabolism were analyzed by ELISA., Key Results: Therapeutic treatment with CYM5520 and LX2931 clearly increased long bone and vertebral bone mass to impressive 3-5 fold over vehicle in osteopenic ovariectomized mice. As expected, lymphopenia was a side effect of LX2931, whereas none occurred with CYM5520. Consistent with an osteoanabolic effect, CYM5520 increased osteoblast number, osteoid surface and alkaline phosphatase area 2-3 fold over vehicle. Plasma concentrations of the osteoanabolic marker procollagen I C-terminal propeptide were also elevated by CYM5520 and LX2931. LX2931 but not yet CYM5520 increased cortical thickness and mechanical strength without affecting mineral density., Conclusion and Implications: Treatment with a pharmacological S1P2 agonist corrected ovariectomy-induced osteopenia in mice by inducing new bone formation thus constituting a novel osteoanabolic approach to osteoporosis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Our previous study demonstrated an indispensable role of Peyer's patches (PPs) for the induction of antigen-specific secretory (S)IgA antibody responses after oral immunization with recombinant Salmonella expressing fragment C of tetanus toxin (rSalmonella-Tox C). In this study, we defined the PP lymphoid structures and immune cells required for the induction of mucosal SIgA antibody responses. Adoptive transfer of mononuclear cells (MNCs) from PPs into PP-deficient (PP-null) mice failed to elicit tetanus toxoid (TT)-specific mucosal immunity. However, when the same PP MNCs were transferred into lethally irradiated PP-normal recipient mice, PP MNCs preferentially emigrated to recipient PPs, leading to PP lymphoid structures and TT-specific SIgA antibody responses. Significantly reduced numbers of TT-specific IgA antibody-forming cells were detected in the mesenteric lymph nodes (MLNs) and intestinal lamina propria of mice when surface expression of the sphingosine 1-phosphate receptor on lymphocytes was inhibited by its agonist FTY720. However, FTY720 treatment did not alter dendritic cell migration or Salmonella dissemination into these tissues. When rSalmonella-Tox C-stimulated CD4+ T cells isolated from PPs, MLNs and the spleen were co-cultured with B cells from these tissues, significantly increased levels of TT-specific IgA antibody responses were exclusively induced in cultures containing PP B cells. Furthermore, surface IgA+ PP B cells produced TT-specific IgA antibody responses in vitro. These findings suggest that PP lymphoid structures and surface IgA+ PP B cells are essential elements for the induction of antigen-specific intestinal SIgA antibody responses to oral Salmonella., (© The Japanese Society for Immunology. 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A decrease in heart rate (HR) is a well-established first-dose effect of sphingosine-1-phosphate subtype 1 receptor (S1P1R) modulators. For compounds with a short half-life ( t 1/2 ), this can be mitigated by gradual up-titration to therapeutic doses, whereas this is not required for compounds with a long t 1/2 due to the less pronounced first-dose-related negative chronotropic effects. Based on this conceptual framework, this mechanistic study investigated whether first-dose HR effects of ponesimod ( t 1/2 ~32 h) can be mitigated by prior administration of cenerimod ( t 1/2 ~415 h). Healthy subjects ( n = 12) were randomly assigned to active or placebo (2:1 ratio). Active treatment consisted of a single dose of 10 mg ponesimod on Day 1, 18, and 37 and multiple-dose administration of 2 mg once daily cenerimod (Day 9-36). Placebos of cenerimod and ponesimod were used as reference treatment. Cardiodynamic parameters were derived from 24 h Holter electrocardiogram (ECG) assessments on Day 1, 9, 10, 18, 36, and 37. Ponesimod (10 mg) alone triggered a transient mean decrease from baseline in hourly mean HR of 17 bpm. In contrast, decreases of 5.0 and 4.8 bpm were observed when ponesimod was given at near half steady-state (Day 18) or steady-state (Day 37) cenerimod, respectively. Hourly mean HR decreased after first administration of cenerimod and placebo was 7.4 and 4.0 bpm, respectively. Treatment with ponesimod and cenerimod alone or in combination was safe and tolerated. First-dose-related negative chronotropic effects of ponesimod were less pronounced when administered after initiation of cenerimod suggesting mitigation of this class-related liability.

Sphingosine-1-phosphate receptor 1 (S1P 1 R), a member of the G protein-coupled receptor (GPCR) family, is an attractive protein target for the treatment of autoimmune diseases, and a diverse array of S1P 1 R agonists have been developed. Rational drug design based on S1P 1 R remains challenging due to the limited information available on the binding mode between S1P 1 R and its agonists. In this work, the active-like state of S1P 1 R was modeled via Gaussian accelerated molecular dynamics (GaMD) based on its inactive form, which was further validated by docking studies with two representative S1P 1 R agonists. Moreover, with the usage of the induced active-like state, the binding mode between S1P 1 R and its agonists was studied through molecular dynamics simulations and MM-GBSA calculations. The results of those studies indicated that four groups of binding site residues were the major contributors to the ligand and receptor interactions. In addition, this model was verified by five chemically similar compounds synthesized in-house and 1145 known S1P 1 R agonists collected from the BindingDB database. The elucidation of the key binding characteristics will further complete the cognition of S1P 1 R, which can guide the rational design of novel S1P 1 R agonists.

Subarachnoid hemorrhage (SAH) results in neurological damage, acute cardiac damage and has a high mortality rate. Immunoresponse in the acute phase after SAH plays a key role in mediating vasospasm, edema, inflammation and neuronal damage. The S1P/S1PR pathway impacts multiple cellular functions, exerts anti-inflammatory and anti-apoptotic effects, promotes remyelination, and improves outcome in several central nervous system (CNS) diseases. RP001 hydrochloride is a novel S1PR agonist, which sequesters lymphocytes within their secondary tissues and prevents infiltration of immune cells into the CNS thereby reducing immune response. In this study, we investigated whether RP001 attenuates neuronal injury after SAH by reducing inflammation. S1PRs, specifically S1PR 1, 3 not only exerts anti-inflammatory effects, but also decreases heart rate and induces atrioventricular conduction abnormalities. Therefore, we also tested whether RP001 treatment of SAH regulates cardiac functional outcome. Male adult C57BL/6 mice were subjected to SAH, and neurological function tests, echocardiography, and immunohistochemical analysis were performed. SAH induces neurological deficits and acute cardiac dysfunction compared to sham control mice. Treatment of SAH with a low-dose of RP001 induces better neurological outcome and cardiac function compared to a high-dose of RP001. Low-dose-RP001 treatment significantly decreases apoptosis, white matter damage, blood brain barrier permeability, microglial/astrocyte activation, macrophage chemokine protein-1, matrix metalloproteinase-9 and NADPH oxidase-2 expression in the brain compared to SAH control mice. Our findings indicate that low-dose of RP001 alleviates neurological damage after SAH, in part by decreasing neuroinflammation., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The pandemic 2009 influenza A H1N1 virus is associated with significant mortality. Targeting S1PR1, which is known to modulate the immune response, provides protection against pathogenic influenza virus. The functional role and molecular mechanism of S1PR1 were analysed by generating inducible endothelial cell-specific S1PR1 knockout mice and assessing the therapeutic efficacy of the selective S1PR1 agonist CYM5442 against acute lung injury (ALI) induced by the 2009 influenza A H1N1 virus. Immune-mediated pulmonary injury is aggravated by the absence of endothelial S1PR1 and alleviated by treatment with CYM-5442, suggesting a protective function of S1PR1 signaling during H1N1 infection. S1PR1 signaling does not affect viral clearance in mice infected with influenza. Mechanistically, the MAPK and NF-kB signaling pathways are involved in the ALI mediated by S1PR1 in infected mice. Combined administration of the S1PR1 agonist CYM-5442 and the antiviral drug oseltamivir provides maximum protection from ALI. Our current study provides insight into the molecular mechanism of S1PR1 mediating the ALI induced by H1N1 infection and indicates that the combination of S1PR1 agonist with antiviral drug could potentially be used as a therapeutic remedy for future H1N1 virus pandemics.

Recently, our research group reported the identification of BMS-986104 (2) as a differentiated S1P 1 receptor modulator. In comparison to fingolimod (1), a full agonist of S1P 1 currently marketed for the treatment of relapse remitting multiple sclerosis (RRMS), 2 offers several potential advantages having demonstrated improved safety multiples in preclinical evaluations against undesired pulmonary and cardiovascular effects. In clinical trials, 2 was found to exhibit a pharmacokinetic half-life ( T 1/2 ) longer than that of 1, as well as a reduced formation of the phosphate metabolite that is required for activity against S1P 1 . Herein, we describe our efforts to discover highly potent, partial agonists of S1P 1 with a shorter T 1/2 and increased in vivo phosphate metabolite formation. These efforts culminated in the discovery of BMS-986166 (14a), which was advanced to human clinical evaluation. The pharmacokinetic/pharmacodynamic (PK/PD) relationship as well as pulmonary and cardiovascular safety assessments are discussed. Furthermore, efficacy of 14a in multiple preclinical models of autoimmune diseases are presented.

Sphingosine-1 phosphate receptor 1 (S1PR1) is expressed by lymphocytes and regulates their egress from secondary lymphoid organs. Innate lymphoid cell (ILC) family has been expanded with the discovery of group 1, 2 and 3 ILCs, namely ILC1, ILC2 and ILC3. ILC3 and ILC1 have remarkable similarity to CD4+ helper T cell lineage members Th17 and Th1, respectively, which are important in the pathology of multiple sclerosis (MS). Whether human ILC subsets express S1PR1 or respond to its ligands have not been studied. In this study, we used peripheral blood/cord blood and tonsil lymphocytes as a source of human ILCs. We show that human ILCs express S1PR1 mRNA and protein and migrate toward S1P receptor ligands. Comparison of peripheral blood ILC numbers between fingolimod-receiving and treatment-free MS patients revealed that, in vivo , ILCs respond to fingolimod, an S1PR1 agonist, resulting in ILC-penia in circulation. Similarly, murine ILCs responded to fingolimod by exiting blood and accumulating in the secondary lymph nodes. Importantly, ex vivo exposure of ILC3 and ILC1 to fingolimod or SEW2871, another S1PR1 antagonist, reduced production of ILC3- and ILC1- associated cytokines GM-CSF, IL-22, IL-17, and IFN-γ, respectively. Surprisingly, despite reduced number of lamina propria-resident ILC3s in the long-term fingolimod-treated mice, ILC3-associated IL-22, IL-17A, GM-CSF and antimicrobial peptides were high in the gut compared to controls, suggesting that its long term use may not compromise mucosal barrier function. To our knowledge, this is the first study to investigate the impact of fingolimod on human ILC subsets in vivo and ex vivo , and provides insight into the impact of long term fingolimod use on ILC populations.

SYL-927, a novel and selective S1P 1 agonist, is transferred to its active phosphate for the regulation of lymphocyte recirculation. This in vitro metabolism study is to elucidate the P450-mediated oxidation pathway of SYL-927 in human liver microsomes (HLMs). The results demonstrated that the ω-1 hydroxylated metabolite SYL-927-M was formed after incubation of SYL-927 with HLMs. Recombinant human CYP1A1 and CYP2J2 can efficiently catalyse SYL-927-M formation, followed by markedly less substrate conversion with CYP1A2, CYP2C19 and CYP2D6. Inhibition studies with chemical inhibitors and antibodies suggested that arachidonic acid, the substrate of CYP2J2, and CYP2J2-specific antibody effectively inhibited the formation of SYL-927-M in HLMs whereas no significant inhibition was observed with the inhibitors for CYP1A1, CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4, demonstrating that CYP2J2 was primarily responsible for the formation of SYL-927-M., (© 2018 John Wiley & Sons, Ltd.)