Sphingosine-1-phosphate Receptor Research Articles

FTY720 Suppresses Pathogenic Retinal Müller Cell Activation and Chronic Progression by Inhibiting the mTOR/NF-κB Signaling Pathway and Regulating Autophagy

Purpose FTY720 is an agonist of the Sphingosine-1-phosphate (S1P) receptor 1, 3, 4, and 5 and a functional antagonist of the S1P1 receptor; it can inhibit the activation of mTOR/NF-κB and has therapeutic potential in inflammatory disease. This study was designed to determine the role of the inflammatory process in diabetic retinopathy and investigate the effect of FTY720 on high glucose (HG)-induced rat retinal Müller cells (rMC-1 cells). Methods In the present study, the role of FTY720 in inhibiting inflammation and its underlying mechanism were investigated. rMC-1 cells were treated without or with HG, FTY720, CQ, or RAP. Cell viability was examined by CCK-8 assay; cell activation was assessed by western blot analysis and IF staining; and cell migration was evaluated by a scratch wound healing assay. The expression of inflammation-associated proteins and autophagy-related proteins was evaluated by transmission electron microscopy, AO staining, MDC-labeled autophagic vacuoles, western blot analysis and ELISA. Results Western blot analysis and IF staining showed that the level of the rMC-1 cell marker GFAP was decreased, while GS was increased in FTY720 groups compared to that in the HG group. The healing assay results showed that compared with HG treatment, FTY720 treatment significantly reduced cell migration. Western blot analysis, ELISA and IF staining showed that compared with HG, FTY720 reduced proinflammatory proteins by inhibiting the mechanistic target of the mTOR/NF-κB signaling pathway and regulating autophagy. Conclusions This study suggests that in an HG-induced rMC-1 cell model, FTY720 significantly inhibited the production of inflammatory cytokines by inhibiting mTOR/NF-κB signaling and regulating autophagy. These findings were associated with a decrease in rMC-1 cell injury, suggesting that FTY720 or related compounds may be valuable modulators of HG-induced retinal injury.

High-Density Lipoprotein Signaling via Sphingosine-1-Phosphate Receptors Safeguards Spontaneously Hypertensive Rats against Myocardial Ischemia/Reperfusion Injury.

High-density lipoprotein (HDL) protects against ischemia/reperfusion (I/R) injury via signaling through scavenger-receptor class B type-I (SR-BI) and sphingosine-1-phosphate receptors (S1PRs). We recently reported that HDL protects the hearts of spontaneously hypertensive rats (SHRs) against I/R injury in an SR-BI-dependent manner. In this study, we examined the role of S1PRs in HDL-induced protection against myocardial I/R injury in hypertensive rats. Hearts from Wistar Kyoto rats (WKYs) and SHRs were subjected to I/R injury using a modified Langendorff system. The hearts were treated with or without HDL in the presence or absence of a receptor- or kinase-specific antagonist. Cardiac hemodynamics and infarct size were measured. Target proteins were analyzed by immunoblotting and ELISA, and nitrite levels were measured using Greis reagent. HDL protected the hearts of WKYs and SHRs against I/R injury. HDL, however, was more protective in WKYs. HDL protection in SHRs required lipid uptake via SR-BI and S1PR1 and S1PR3 but not S1PR2. The hearts from SHRs expressed significantly lower levels of S1PR3 than the hearts from WKYs. HDL differentially activated mediators of the SAFE and RISK pathways in WKYs and SHRs and resulted in nitric oxide generation. Blockage of these pathways abrogated HDL effects. HDL protects against myocardial I/R injury in normotensive and hypertensive rats, albeit to varying degrees. HDL protection in hearts from hypertensive rodents involved SR-BI-mediated lipid uptake coupled with signaling through S1PR1 and S1PR3. The extent of HDL-induced cardiac protection is directly proportional to S1PR3 expression levels. Mechanistically, the safeguarding effects of HDL involved activation of the SAFE and RISK pathways and the generation of nitric oxide.

Myeloid Cells and Sphingosine-1-Phosphate Are Required for TCRαβ Intraepithelial Lymphocyte Recruitment to the Colon Epithelium.

Intraepithelial lymphocytes (IELs) are T cells important for the maintenance of barrier integrity in the intestine. Colon IELs are significantly reduced in both MyD88-deficient mice and those lacking an intact microbiota, suggesting that MyD88-mediated detection of bacterial products is important for the recruitment and/or retention of these cells. Here, using conditionally deficient MyD88 mice, we show that myeloid cells are the key mediators of TCRαβ+ IEL recruitment to the colon. Upon exposure to luminal bacteria, myeloid cells produce sphingosine-1-phosphate (S1P) in a MyD88-dependent fashion. TCRαβ+ IEL recruitment may be blocked using the S1P receptor antagonist FTY720, confirming the importance of S1P in the recruitment of TCRαβ+ IELs to the colon epithelium. Finally, using the TNFΔARE/+ model of Crohn's-like bowel inflammation, we show that disruption of colon IEL recruitment through myeloid-specific MyD88 deficiency results in reduced pathology. Our results illustrate one mechanism for recruitment of a subset of IELs to the colon.

Synthesis, radiosynthesis and biochemical evaluation of fluorinated analogues of sphingosine-1-phosphate receptor 3 specific antagonists using PET

Sphingosine-1-phosphate and its receptors (S1PRs) are involved in several diseases such as auto immunity, inflammation and cardiovascular disorders. The S1P analogue fingolimod (Gilenya®) is currently in use for the treatment of relapsing multiple sclerosis. S1PRs are also promising targets for clinical molecular imaging in vivo. The organ distribution of individual S1PRs can be potentially achieved by using S1PR subtype-specific (radiolabeled) chemical probes. Here, we report our efforts on synthesis and in vivo potency determination of new ligands for the S1P receptor 3 (S1P3) based on the S1P3 antagonist TY-52156 and in validation of a potential imaging tracer in vivo using Positron Emission Tomography (PET) after 18F-labelling. A p-fluorophenyl derivative exhibited excellent S1P3 antagonist activity in vitro, good serum stability, and medium lipophilicity. In vivo biodistribution experiments using 18F-PET exhibited significant uptake in the myocardium suggesting potential applications in cardiac imaging.

Abstract 2659: Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response

Abstract Introduction: Immunotherapy has revolutionized the landscape of cancer treatment; however, to date, only limited aspects of the tumor-immune interaction have been exploited successfully, e.g., relief of immune checkpoints, increase of tumor-associated antigen presentation. Although it is well-known that trafficking and entry of T cells from the circulation into tumors is often impaired, leading to an immune “cold”, checkpoint inhibitor-refractory state, there are no available therapies that address this important aspect of antitumor immunity. Sphingosine-1-phosphate (S1P) is a pleiotropic, bioactive lipid that has been shown to regulate lymphocyte egress from lymph nodes through its interaction with one of its several receptors, S1P1. Clinically, tumor expression levels of the sphingosine-1-phosphate-producing enzyme sphingosine kinase 1 (SPHK1) strongly and negatively correlates with survival of melanoma patients after immune checkpoint inhibitor (ICI) therapy, and pre-clinically correlates with reduced tumor infiltrating lymphocytes (TILs). We therefore hypothesize that aberrant S1P signaling in tumors and in their draining lymph nodes (TDLN) causes immune exclusion by impeding lymphocyte egress from the TDLN, ultimately restricting immune cell infiltration of tumors. Methods: We tested the therapeutic efficacy of an engineered S1P-degrading enzyme, S1P lyase (S1PL), on established (~100mm^3) syngeneic B16, CT26, and MC38 murine tumors. Using flow cytometry, we performed immune profiling of systemic, TDLN, and intratumoral T cell populations, assessing both activation status and expression of the S1P1 receptor. Results: Treatment of tumor-bearing animals with S1PL depleted systemic S1P, and dramatically increased both trafficking of lymphocytes out of TDLNs and T cell infiltration of tumors relative to control-treated animals. Mice treated with S1PL evinced a higher proportion of S1P1+ circulating and intratumoral T cells relative to control-treated mice, suggesting that the increase in liberation of T cells from the TDLN was effected through the S1P/S1P1 axis. This increased lymphocyte trafficking correlated with strong inhibition of tumor growth when S1PL was administered as a single agent. Conclusions: Therapeutic administration of S1PL may represent an attractive strategy for modulating lymphocyte trafficking to improve the efficacy of therapies that rely on the adaptive immune system to exert antitumor activity. This approach may be particularly potent in settings wherein tumor-mediated immune exclusion is a dominant method of immune escape. Citation Format: Alessandra Araujo, Max Rodnick-Smith, Ranya Al-Khaledy, George Georgiou, Mark Badeaux, Everett Stone. Therapeutic administration of an engineered sphingosine-1-phosphate lyase improves T cell trafficking to tumors and enhances antitumor response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2659.

Shuangshen Granules Suppress Myeloid-derived Suppressor Cell-mediated Lung Premetastatic Niche Development by Targeting Sphingosine-1-Phosphate Receptor-1/Signal Transducer, Activator of Transcription 3 Signaling

Abstract Background: Shuangshen granules (SSGs) are extensively utilized for the treatment of lung cancer in China and have been reported to possess tumor-protective and anti-metastatic effects. Therefore, it is crucial to understand the precise mechanism. Building upon the findings of our previous study, the objective of the present study was to explore the impact of SSGs on the sphingosine-1-phosphate receptor-1 (S1PR1)/signal transducer and activator of transcription 3 (STAT3) axis, as well as the recruitment of myeloid-derived suppressor cells (MDSCs) during the formation of the premetastatic niches (PMNs). Methods: In a mouse xenograft model utilizing Lewis lung carcinoma (LLC) cells that express green fluorescent protein (GFP), the initiation of lung metastasis was monitored every three days until day 35 following transplantation. Lung metastasis, MDSC recruitment, the expression of PMN and S1PR1/STAT3 axis biomarkers, as well as the blood levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor-β (TGF-β) were assessed in the SSG treatment and control groups. Results: The LLC cells did not reach the lung until 14–17 days following subcutaneous implantation, which was concurrent with the formation of lung PMNs. SSG significantly postponed the initiation of lung metastasis and reduced the recruitment of MDSCs to the lung PMNs. SSG also suppressed the S1PR1/STAT3 axis in tumor tissues, bone marrow, and lung PMNs. Additionally, SSG suppressed the blood levels of GM-CSF and TGF-β, as well as the PMN markers, matrix metalloproteinase-9 and versican. Conclusion: Our findings suggested that SSG suppressed the development of MDSC-mediated PMNs by inhibiting the S1PR1/STAT3 axis, consequently postponing the initiation of lung metastasis.

The β-catenin C terminus links Wnt and sphingosine-1-phosphate signaling pathways to promote vascular remodeling and atherosclerosis.

Canonical Wnt and sphingosine-1-phosphate (S1P) signaling pathways are highly conserved systems that contribute to normal vertebrate development, with key consequences for immune, nervous, and cardiovascular system function; despite these functional overlaps, little is known about Wnt/β-catenin-S1P cross-talk. In the vascular system, both Wnt/β-catenin and S1P signals affect vessel maturation, stability, and barrier function, but information regarding their potential coordination is scant. We report an instance of functional interaction between the two pathways, including evidence that S1P receptor 1 (S1PR1) is a transcriptional target of β-catenin. By studying vascular smooth muscle cells and arterial injury response, we find a specific requirement for the β-catenin carboxyl terminus, which acts to induce S1PR1, and show that this interaction is essential for vascular remodeling. We also report that pharmacological inhibition of the β-catenin carboxyl terminus reduces S1PR1 expression, neointima formation, and atherosclerosis. These findings provide mechanistic understanding of how Wnt/β-catenin and S1P systems collaborate during vascular remodeling and inform strategies for therapeutic manipulation.

Peripheral oedema as an adverse effect of treatment of secondary progressive multiple sclerosis with siponimod: A case series

Siponimod is a sphingosine 1-phosphate receptor (S1P) modulator used to treat secondary progressive multiple sclerosis (SPMS). We report 3 SPMS patients treated with siponimod who developed new or worsening peripheral oedema soon after commencing treatment. In one case, peripheral oedema resulted in immobility. Siponimod-related peripheral oedema deserves wider recognition due to the potential for morbidity and over-investigation. Clinicians should assess for pre-existing oedema and coexisting conditions that may predispose to developing peripheral oedema prior to commencing siponimod.

Is myeloid-derived growth factor a ligand of the sphingosine-1-phosphate receptor 2?

Secretory myeloid-derived growth factor (MYDGF) exerts beneficial effects on organ repair, probably via a plasma membrane receptor; however, the identity of the expected receptor has remained elusive. In a recent study, MYDGF was reported as an agonist of the sphingosine-1-phosphate receptor 2 (S1PR2), an A-class G protein-coupled receptor that mediates the functions of the signaling lipid, sphingosine-1-phosphate (S1P). In the present study, we conducted living cell-based functional assays to test whether S1PR2 is a receptor for MYDGF. In the NanoLuc Binary Technology (NanoBiT)-based β-arrestin recruitment assay and the cAMP-response element (CRE)-controlled NanoLuc reporter assay, S1P could efficiently activate human S1PR2 overexpressed in human embryonic kidney (HEK) 293T cells; however, recombinant human MYDGF, overexpressed either from Escherichia coli or HEK293 cells, had no detectable effect. Thus, the results demonstrated that human MYDGF is not a ligand of human S1PR2. Considering the high conservation of MYDGF and S1PR2 in evolution, MYDGF is also probably not a ligand of S1PR2 in other vertebrates.

SAA1 regulated by S1P/S1PR1 promotes the progression of ESCC via β-catenin activation

Serum amyloid A1 (SAA1), an inflammation-related molecule, is associated with the malignant progression of many tumors. This study aimed to investigate the role of SAA1 in the progression of esophageal squamous cell carcinoma (ESCC) and its molecular mechanisms. The expression of SAA1 in ESCC tissues and cell lines was analyzed using bioinformatics analysis, western blotting, and reverse transcription-quantitative PCR (RT‒qPCR). SAA1-overexpressing or SAA1-knockdown ESCC cells were used to assess the effects of SAA1 on the proliferation, migration, apoptosis of cancer cells and the growth of xenograft tumors in nude mice. Western blotting, immunofluorescence and RT‒qPCR were used to investigate the relationship between SAA1 and β-catenin and SAA1 and sphingosine 1-phosphate (S1P)/sphingosine 1-phosphate receptor 1 (S1PR1). SAA1 was highly expressed in ESCC tissues and cell lines. Overexpression of SAA1 significantly promoted the proliferation, migration and the growth of tumors in nude mice. Knockdown of SAA1 had the opposite effects and promoted the apoptosis of ESCC cells. Moreover, SAA1 overexpression promoted the phosphorylation of β-catenin at Ser675 and increased the expression levels of the β-catenin target genes MYC and MMP9. Knockdown of SAA1 had the opposite effects. S1P/S1PR1 upregulated SAA1 expression and β-catenin phosphorylation at Ser675 in ESCC cells. In conclusion, SAA1 promotes the progression of ESCC by increasing β-catenin phosphorylation at Ser675, and the S1P/S1PR1 pathway plays an important role in its upstream regulation.

The Effects of the S1P Receptor Agonist Fingolimod (FTY720) on Central and Peripheral Myelin in Twitcher Mice.

Krabbe's disease (KD) is caused by mutations in the lysosomal enzyme galactocerebrosidase and is associated with psychosine toxicity. The sphingosine 1-phosphate receptor (S1PR) agonist fingolimod (FTY720) attenuates psychosine-induced cell death of human astrocytes, demyelination in cerebellar slices, as well as demyelination in the central nervous system of twitcher mice. Psychosine also accumulates in the peripheral nervous system in twitcher mice; however, effects of fingolimod on this peripheral myelin have not been examined. The aim of this study was to investigate the effects of fingolimod administration on peripheral and central markers of myelination. Here, we report that fingolimod administration (1 mg/kg/day) from postnatal day 5 (PND) onwards did not alter peripheral demyelination in the sciatic nerve of twitcher mice, despite significantly reducing myelin debris, glial reactivity, and neuronal damage in the cerebellum. We also find fingolimod administration improves twitching and mobility scores in twitcher mice. Importantly, we find that fingolimod significantly increases the lifespan of twitcher mice by approximately 5 days. These findings suggest differential effects of fingolimod on peripheral and central neuropathy in twitcher mice, which may explain its modest efficacy on behavior and lifespan.

Differences in collateral vessel formation after experimental retinal vein occlusion in spontaneously hypertensive rats and wild-type rats

ObjectiveRetinal vein occlusion (RVO) can lead to visual impairment, but the development of collateral vessels can sometimes mitigate significant damage. This study aimed to investigate the relationship between collateral vessels and hypertension, the most common underlying condition associated with RVO, by comparing spontaneously hypertensive rats (SHRs) and wild-type Wister rats (WWRs). We also examined the differences between WWRs and SHRs in terms of sphingosine 1-phosphate receptor 1 (S1PR1) expression and its product nitric oxide synthase 3 (NOS3) expression, which are involved in the formation of collateral vessels after vascular occlusion. MethodsLaser photocoagulation (PC) was used to occlude one randomly selected retinal vein in WWRs and SHRs, and the area surrounding the occluded vessel was examined using optical coherence tomography angiography. If reperfusion of the occluded vessel occurred within 2 weeks, the vessel was re-occluded repeatedly by PC. The number of eyes with successfully occluded vessels accompanied by collateral vessels was recorded. Then, WWRs and SHRs were divided into the following four groups: 1) control (no treatment), 2) vehicle (20% DMSO), 3) S1PR1 agonist (2 mg/mL SEW2871), and 4) S1PR1 antagonist (0.25 mg/mL VPC 23019) groups. The drugs were administered intravitreally in all groups except the control. The number of laser shots required for successful RVO was recorded. Histological evaluation and quantitative real-time PCR of S1PR1 and NOS3 were performed to elucidate the mechanisms underlying collateral vessel formation. ResultsThe proportion of eyes achieving successful vein occlusion was lower in SHRs (4/12 eyes, 33.3%) than in WWRs (8/10 eyes, 80%, p = 0.043). NOS3 expression at 6 h after PC was significantly higher in WWRs than in SHRs (p = 0.021). In WWRs treated with SEW2871, vein occlusion failed in 7 of 10 eyes (70%). The expression of NOS3 was significantly higher in the SEW2871 treatment group than in the untreated group (p < 0.001). Furthermore, NOS3 expression was significantly higher after SEW2871 treatment in WWRs than in SHRs (p = 0.011). ConclusionIn hypertensive environments, collateral vessels are less likely to develop, and S1PR1 may be involved in this phenomenon.

A novel model to study mechanisms of cholestasis in human cholangiocytes reveals a role for the SIPR2 pathway.

Ductular reactivity is central to the pathophysiology of cholangiopathies. Mechanisms underlying the reactive phenotype activation by exogenous inflammatory mediators and bile acids are poorly understood. Using human extrahepatic cholangiocyte organoids (ECOs) we developed an injury model emulating the cholestatic microenvironment with exposure to inflammatory mediators and various pathogenic bile acids. Moreover, we explored roles for the bile acid activated Sphingosine-1-phosphate receptor 2 (S1PR2) and potential beneficial effects of therapeutic bile acids UDCA and norUDCA. Synergistic exposure to bile acids (taurocholic acid, glycocholic acid, glycochenodeoxycholic acid) and TNF-α for 24 hours induced a reactive state as measured by ECO diameter, proliferation, lactate dehydrogenase activity and reactive phenotype markers. While NorUDCA and UDCA treatments given 8 hours after injury induction both suppressed reactive phenotype activation and most injury parameters, proliferation was improved by NorUDCA only. Extrahepatic cholangiocyte organoid stimulation with S1PR2 agonist sphingosine-1-phosphate reproduced the cholangiocyte reactive state and upregulated S1PR2 downstream mediators; these effects were suppressed by S1PR2 antagonist JET-013 (JET), downstream mediator extracellular signal-regulated kinase 1/2 inhibitor, and by norUDCA or UDCA treatments. JET also partially suppressed reactive phenotype after bile acid injury. We developed a novel model to study the reactive cholangiocyte state in response to pathological stimuli in cholestasis and demonstrated a contributory role of S1PR2 signaling in both injury and NorUDCA/UDCA treatments. This model is a valuable tool to further explore the pathophysiology of human cholangiopathies.

Expression and prognostic role of sphingosine 1-phosphate receptor 4 (S1PR4) as a biomarker of skin cutaneous melanoma

BackgroundSkin cutaneous melanoma (SKCM) is one of the most lethal skin malignancies worldwide. Sphingosine 1-phosphate (S1P) regulates tumor cells through S1P receptors (S1PRs). Unlike S1PR1/2/3/5, whose anti-apoptotic effects have been widely studied, the regulatory effect of S1PR4 on tumors has not been studied extensively. In this study, we aimed to investigate the correlation between S1PR4 expression and survival, clinical manifestations, tumor microenvironment, and immune infiltration in patients with SKCM. ResultsLow S1PR4 expression was associated with poor prognosis in patients with SKCM. Patients in the high-expression group had significantly longer disease survival and progression-free survival than those in the low-expression group. ConclusionHigh S1PR4 expression was highly associated with better prognosis and milder clinical manifestations; thus, S1PR4 may be used as a prognostic marker to help physicians monitor patients with SKCM.

CD4+ CD11b+ T cells infiltrate and aggravate the traumatic brain injury depending on brain-to-cervical lymph node signaling.

We aim to identify the specific CD4+ T-cell subtype influenced by brain-to-CLN signaling and explore their role during the acute phase of traumatic brain injury (TBI). Cervical lymphadenectomy or cervical afferent lymphatic ligation was performed before TBI. Cytokine array and western blot were used to detect cytokines, while the motor function was assessed using mNss and rotarod test. CD4+ T-cell subtypes in blood, brain, and CLNs were analyzed with Cytometry by time-of-flight analysis (CyTOF) or fluorescence-activated cell sorting (FACS). Brain edema and volume changes were measured by 9.4T MRI. Neuronal apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Cervical lymphadenectomy and ligation of cervical lymphatic vessels resulted in a decreased infiltration of CD4+ T cells, specifically CD11b-positive CD4+ T cells, within the affected region. The population of CD4+ CD11b+ T cells increased in ligated CLNs, accompanied by a decrease in the average fluorescence intensity of sphingosine-1-phosphate receptor-1 (S1PR1) on these cells. Administration of CD4+ CD11b+ T cells sorted from CLNs into the lateral ventricle reversed the attenuated neurologic deficits, brain edema, and lesion volume following cervical lymphadenectomy. The infiltration of CD4+ CD11b+ T cells exacerbates secondary brain damage in TBI, and this process is modulated by brain-to-CLN signaling.

How do sphingosine-1-phosphate affect immune cells to resolve inflammation?

Inflammation is an important immune response of the body. It is a physiological process of self-repair and defense against pathogens taken up by biological tissues when stimulated by damage factors such as trauma and infection. Inflammation is the main cause of high morbidity and mortality in most diseases and is the physiological basis of the disease. Targeted therapeutic strategies can achieve efficient toxicity clearance at the inflammatory site, reduce complications, and reduce mortality. Sphingosine-1-phosphate (S1P), a lipid signaling molecule, is involved in immune cell transport by binding to S1P receptors (S1PRs). It plays a key role in innate and adaptive immune responses and is closely related to inflammation. In homeostasis, lymphocytes follow an S1P concentration gradient from the tissues into circulation. One widely accepted mechanism is that during the inflammatory immune response, the S1P gradient is altered, and lymphocytes are blocked from entering the circulation and are, therefore, unable to reach the inflammatory site. However, the full mechanism of its involvement in inflammation is not fully understood. This review focuses on bacterial and viral infections, autoimmune diseases, and immunological aspects of the Sphks/S1P/S1PRs signaling pathway, highlighting their role in promoting intradial-adaptive immune interactions. How S1P signaling is regulated in inflammation and how S1P shapes immune responses through immune cells are explained in detail. We teased apart the immune cell composition of S1P signaling and the critical role of S1P pathway modulators in the host inflammatory immune system. By understanding the role of S1P in the pathogenesis of inflammatory diseases, we linked the genomic studies of S1P-targeted drugs in inflammatory diseases to provide a basis for targeted drug development.

Reversible oxidation/reduction steps in the metabolic degradation of the glycerol side chain of the S1P1 modulator ponesimod

1. Ponesimod is a selective modulator of the sphingosine 1-phosphate receptor 1 (S1P1) approved for the treatment of active relapsing forms of multiple sclerosis. The chemical structure of ponesimod contains a glycerol side chain which is the major target of drug metabolism in humans. 2. The two major metabolic pathways give the acids M12 (-OCH2CH(OH)COOH) and M13 (-OCH2COOH). While the former results from oxidation of the terminal alcohol, the mechanism yielding the chain-shortened acid M13 is less obvious. A detailed mechanistic study with human liver microsomes and hepatocytes using ponesimod, M12 and some of the suspected intermediates revealed an unexpectedly complex pattern of enzyme-mediated and chemical reactions. 3. Metabolic pathways for both acids were not independent and several of the transformations were reversible, depending on reaction conditions. Formation of M13 occurred either via initial oxidation of the secondary alcohol, or as a downstream process starting from M12. 4. The phenol metabolite M32 was produced as part of several pathways. Control experiments at various pH values and in the absence of metabolizing enzymes support the conclusion that its formation resulted from chemical degradation rather than from metabolic processes.

Siponimod Attenuates Neuronal Cell Death Triggered by Neuroinflammation via NFκB and Mitochondrial Pathways.

Multiple sclerosis (MS) is the most common autoimmune demyelinating disease of the central nervous system (CNS), consisting of heterogeneous clinical courses varying from relapsing-remitting MS (RRMS), in which disability is linked to bouts of inflammation, to progressive disease such as primary progressive MS (PPMS) and secondary progressive MS (SPMS), in which neurological disability is thought to be linked to neurodegeneration. As a result, successful therapeutics for progressive MS likely need to have both anti-inflammatory and direct neuroprotective properties. The modulation of sphingosine-1-phosphate (S1P) receptors has been implicated in neuroprotection in preclinical animal models. Siponimod/BAF312, the first oral treatment approved for SPMS, may have direct neuroprotective benefits mediated by its activity as a selective (S1P receptor 1) S1P1 and (S1P receptor 5) S1P5 modulator. We showed that S1P1 was mainly present in cortical neurons in lesioned areas of the MS brain. To gain a better understanding of the neuroprotective effects of siponimod in MS, we used both rat neurons and human-induced pluripotent stem cell (iPSC)-derived neurons treated with the neuroinflammatory cytokine tumor necrosis factor-alpha (TNF-α). Cell survival/apoptotic assays using flow cytometry and IncuCyte live cell analyses showed that siponimod decreased TNF-α induced neuronal cell apoptosis in both rat and human iPSCs. Importantly, a transcriptomic analysis revealed that mitochondrial oxidative phosphorylation, NFκB and cytokine signaling pathways contributed to siponimod's neuroprotective effects. Our data suggest that the neuroprotection of siponimod/BAF312 likely involves the relief of oxidative stress in neuronal cells. Further studies are needed to explore the molecular mechanisms of such interactions to determine the relationship between mitochondrial dysfunction and neuroinflammation/neurodegeneration.

Sphingosine 1-phosphate receptor modulators in multiple sclerosis treatment: A practical review.

Four sphingosine 1-phosphate (S1P) receptor modulators (fingolimod, ozanimod, ponesimod, and siponimod) are approved by the US Food and Drug Administration for the treatment of multiple sclerosis. This review summarizes efficacy and safety data on these S1P receptor modulators, with an emphasis on similarities and differences. Efficacy data from the pivotal clinical trials are generally similar for the four agents. However, because no head-to-head clinical studies were conducted, direct efficacy comparisons cannot be made. Based on the adverse event profile of S1P receptor modulators, continued and regular monitoring of patients during treatment will be instructive. Notably, the authors recommend paying attention to the cardiac monitoring guidelines for these drugs, and when indicated screening for macular edema and cutaneous malignancies before starting treatment. To obtain the best outcome, clinicians should choose the drug based on disease type, history, and concomitant medications for each patient. Real-world data should help to determine whether there are meaningful differences in efficacy or side effects between these agents.

Fine-tuning activation specificity of G-protein-coupled receptors via automated path searching

Physics-based simulation methods can grant atomistic insights into the molecular origin of the function of biomolecules. However, the potential of such approaches has been hindered by their low efficiency, including in the design of selective agonists where simulations of myriad protein-ligand combinations are necessary. Here, we describe an automated input-free path searching protocol that offers (within 14 d using Graphics Processing Unit servers) a minimum free energy path (MFEP) defined in high-dimension configurational space for activating sphingosine-1-phosphate receptors (S1PRs) by arbitrary ligands. The free energy distributions along the MFEP for four distinct ligands and three S1PRs reached a remarkable agreement with Bioluminescence Resonance Energy Transfer (BRET) measurements of G-protein dissociation. In particular, the revealed transition state structures pointed out toward two S1PR3 residues F263/I284, that dictate the preference of existing agonists CBP307 and BAF312 on S1PR1/5. Swapping these residues between S1PR1 and S1PR3 reversed their response to the two agonists in BRET assays. These results inspired us to design improved agonists with both strong polar head and bulky hydrophobic tail for higher selectivity on S1PR1. Through merely three in silico iterations, our tool predicted a unique compound scaffold. BRET assays confirmed that both chiral forms activate S1PR1 at nanomolar concentration, 1 to 2 orders of magnitude less than those for S1PR3/5. Collectively, these results signify the promise of our approach in fine agonist design for G-protein-coupled receptors.